USER GUIDE
ProteographTM Analysis Suite
seer.bio

Notice
© 2021 Seer, Inc. All rights reserved. Information in this document is subject to change without notice. Seer assumes no responsibility for any errors or omissions in this document. Duplication and/or reproduction of all or any portion of this document without the express written consent of Seer is strictly forbidden. Seer does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.
Nothing contained herein shall constitute any warranty, express or implied, as to the performance of any products described herein, or any warranties of merchantability, fitness for a particular purpose or non-infringement. The use of products described herein is subject to certain restrictions as set forth in the applicable terms and conditions of sale or license accompanying the purchase of such product and any written agreements between Seer and user. Seer may refer to products or services offered by other companies by their brand names or company names solely for clarity and does not claim any rights in those third party marks or names. All products and services described herein are intended FOR RESEARCH USE ONLY and NOT FOR USE IN DIAGNOSTIC PROCEDURES.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).
Failure to completely read and explicitly follow all of the instructions contained herein may result in damage to the product(s), injury to persons, including to users or others, and damage to other property, and will void any warranty applicable to the product(s). Seer does not assume any liability arising out of the improper use of the product(s) described herein (including parts thereof or software).
BUYER IS NOT LICENSED OR AUTHORIZED TO, AND AGREES NOT TO, USE ANY SEER PRODUCT FOR ANY CLINICAL OR DIAGNOSTIC PURPOSES.
Notice
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 2

Revision history
DOCUMENT DATE
CF-1003 B October (PAS version 1.5) 2021
DESCRIPTION OF CHANGE
Per Proteograph Analysis Suite Release Notes (CF-1018 A)
New analyses to visualize differences in peptide/protein group intensities between samples and groups
• New interactive table containing peptide/protein group intensities across samples/groups
• New heatmap plot for visualizing peptide/protein group intensities across samples/groups
New analysis to identify peptide/protein groups with significantly different intensities between groups (e.g., healthy vs. disease samples)
• Added new “Group analysis” sections to ‘Analysis’ section
• Guided comparison setup providing data filtering, normalization, imputation,
and statistical test options
• Toggle for viewing only ‘Significant Peptides/Proteins’ in dataset
• Interactive plots and tables for exploratory expression analysis of significantly
regulated targets with dynamic fold-change and p-value options
• New group analysis plots to visualize peptides/protein groups with statistically
different intensities
– Volcano plot – fold-change vs. statistical significance with coloring options
to highlight proteins-of-interest
– Coverage viewer – Amino acid sequence coverage by peptides display,
including PTM detection
– Clustered heatmap – visualizing peptide/protein group intensities across
samples/groups with rows (peptide/protein groups) and columns (samples)
ordered using hierarchical clustering
– Protein-protein interaction – Visualization of PPIs (STRING db) of
significant proteins
– Intensity box plots – Compare significant peptide/protein group intensities
between groups
Changes to plot visualizations
• Added ability to export plot source data to .csv files
• Improved plot rendering speeds
• Added user preferences to modify plots
New analysis protocols
• Integration of DIA-NN database search engine
– Pre-installed DIA-NN library-based protocol (DIANN – System Provided
DIA Protocol)
– Expanded Seer human plasma spectral library file (4,011 protein groups;
62,687 precursors)
– Optional in silico predicted library-free protocol
Revision history
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 3

Revision history
DOCUMENT
DATE
DESCRIPTION OF CHANGE
Changes to results summary
• Simplified peptide and protein group results tables
– Panel level summary
– NP level summary
Changes to help content
• Updated user guide
• Indexed online help
• Tooltips
New data file management
• New AutoUploader application allows automatic data transfer from LC-MS system to PAS account
Initial release
CF-1003 A (PAS version 1.0)
May 2021
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 4

Contents
Notice
Revision history Contents
Chapter 1 Welcome to PAS
Access the PAS application
Tour of the PAS application Sidebar menu
Top bar and User Account menu Work with PAS tables
Show or hide table columns
Add custom columns
Sort a table
Filter a table; search for a specific item
Control how many items are shown per table page Navigate among table pages
Access item details and/or sub-items Set color preferences in PAS graphs
Get help with PAS
Manage your user profile
Input files
Plate map file format
Sample description file format
Output files
Quality control Application settings
Change the appearance of the user interface Configure application settings
Configure two-factor authentication for yourself Configure default display settings for analysis results
User management
Users and Permissions page Add a user
Edit a user
Delete a user
Chapter 2 Analysis Setup
Analysis setup workflows
Setup workflow: Add a plate
Add one or more MS data files
Add the plate map file
Specify plate information
Add a sample description file (optional) Add the samples to a project
Start the analysis
Setup workflow: Link to a plate
Review the MS data files to be linked a plate Add the plate map file
2 3 5
9
10 11 11 11 12 12 12 13 13 13 13 13 14 14 14 15 15 16 17 18 19 19 19 19 20 21 21 21 22 22
23
24
25
25
25
26
26
26
27
28
28
28
Contents
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
5

Specify plate information
Add a sample description file (optional) Add the samples to a project
Start the analysis
Setup workflow: Select samples or controls for analysis Analyze selected samples
Analyze controls only
Chapter 3 Analysis Results
Access analysis results
Review analysis results Data visualization Analysis Summary tab
Open an analysis’s Analysis Summary tab Summary section
Protein Group Counts section
Set Protein Group Counts preferences Peptide Counts section
Set Peptide Counts preferences
Distribution of Detected Proteins in Plasma Proteome section
Set Distribution of Detected Proteins in Plasma Proteome preferences Protein Group Overlap Sets section
Set Protein Group Overlap Sets preferences Sample Comparability section
Set Sample Comparability preferences Analysis Metrics tab
Open an analysis’s Analysis Metrics tab Intensities section
Set Intensities preferences Plate Map Grid section
Set Plate Map Grid preferences Lamppost Proteins’ Concentration section
Set Lamppost Proteins’ Concentration preferences PCA Analysis section
Change the PCA Analysis color scheme
Peptide Counts Distribution and Protein Group Counts Distribution sections
Protein Group Counts of Nanoparticles and Peptide Counts of Nanoparticles sections
Set Protein Group Counts of Nanoparticles and Peptide Counts of Nanoparticles preferences Hierarchical Clustering section
QC Charts tab
Open and filter an analysis’s QC Charts tab
Set QC Charts preferences
Manage user-defined and calculated control limits
Define and use user-defined control limits Define and use calculated control limits Delete a calculated control limit date range Exclude a sample
Annotate control charts Add an annotation Edit an annotation
29 29 29 30 31 31 32
33
34 35 36 37 37 37 37 38 38 39 39 40 40 41 42 42 44 44 44 45 45 46 46 47 48 48 49 50 51 52 53 54 55 55 56 56 56 56 57 57 57
Contents
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
6

Delete an annotation
Generate reports of control results
Download summarized control results
Generate a detailed PDF report of control results
Analysis Output Files tab
Chapter 4 Analysis Interpretation
Open an analysis’s Group Analysis tab
Export raw data to a file
View a raw protein group data heatmap Set up and start a group analysis
Specify the quantitation level and grouping of samples Filter out (exclude) samples and groups
Filter out (exclude) proteins or peptides
Normalize values and impute sparse or missing values Select the statistical test and the groups to compare
Group Analysis tab Raw Data section
Clustered Heatmap section Volcano section
Coverage section
Filtered PPI Network section Enrichment section
Box Plots section
Chapter 5 Data Management
Plates and samples
Plates page Review a plate
Edit a plate
Delete a plate Plate Samples section
Add samples to an existing plate Add sample descriptions
Edit sample information
Delete a sample from a plate
Plate Grid section
Edit or delete samples in wells
Upload a custom file containing sample information
Projects
Projects page
Add a project
Delete a project Sample List section
Add samples to a project Analyses
Analyses page
Download an analysis log
Download an analysis’s protein groups and peptides results Edit an analysis
57 57 57 58 59
60
61 62 63 64 64 64 64 65 65 66 66 68 69 70
71 72 73
75
76 76 77 77 77 78 79 80 80 81 81 81 82 83 83 84 84 84 85 86 86 87 87 87
Contents
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
7

Delete one or more analyses
MS data files
Data Files page
Create a folder
Upload MS data files
Move MS data files
Rename MS data files or folders
Delete MS data files or folders
Download and install the Seer AutoUploader application
Analysis protocols Protocols page
View an analysis protocol’s search engine parameters Upload an analysis protocol
Copy an analysis protocol
Download an analysis protocol
Delete an analysis protocol
Glossary
Index
Technical Support
Contact Information
Telephone
88 89 89 90 90 90 90 91 91 92 92 93 93 94 94 94
95
99
102
102 102
Contents
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
8

Chapter 1
Welcome to PAS
Introduces you to the Seer ProteographTM Analysis Suite (PAS) software application, part of the Seer ProteographTM Product Suite.
With this dedicated software solution, you can process, analyze, and visualize proteomics data sets generated by liquid chromatography-mass spectrometry (LC-MS). PAS includes an integrated search engine for identification and annotation of LC-MS data.

Chapter 1 Welcome to PAS Access the PAS application
Access the PAS application
To access the PAS application, point your web browser to pas.seer.software. Compatible browsers include Chrome, Chromium, Edge, Firefox, and Safari.
When prompted to log in, enter your username and password. In the future, if you configure two-factor authentication, you will also need to enter a code generated by an authenticator app on your mobile device. (For details for setting up two-factor authentication, see Configure two-factor authentication for yourself (page 19).)
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 10

Chapter 1 Welcome to PAS
Tour of the PAS application
Tour of the PAS application
The PAS application window has several main parts.
Sidebar menu
Use the sidebar menu to access PAS functionality and data.
A Plates — For importing sample metadata from the Proteograph Assay
method and associated MS data files. See Plates and samples (page 76).
B Projects — For grouping Proteograph Assay plates at the experimental
project level and submitting data analysis jobs. See Projects (page 83).
C Analyses — For accessing data analysis jobs, data analysis results, and data
visualizations. See Analyses (page 86).
D Data Files — For accessing the repository of MS data files and managing
your data. See MS data files (page 89).
E Analysis Protocol — For accessing a collection of database search analysis
protocols. See Analysis protocols (page 92).
F Users — (Admin only) For viewing, adding, editing, and deleting users. See
User management (page 21).
G Help — For accessing PAS documentation including the user guide (PDF),
web-based Help system, and videos. See Get help with PAS (page 14).
Top bar and User Account menu
The top bar of the PAS application window has just one component, the User Account menu, at the far right. To open the menu, select your username.
A B
C D
E
Sidebar menu
Button to expand and collapse the sidebar menu
Top bar
User Account menu, accessible from your username
Page with table items. For general table techniques, see Work with PAS tables (next page).
NOTE
When the sidebar menu is collapsed, the top bar — including your username — is hidden.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 11

Chapter 1 Welcome to PAS
Tour of the PAS application
A B C
D
User Profile — For managing your user information, such as your telephone number. See Manage your user profile (page 14).
Appearance — For selecting a light or dark theme for the PAS user interface. See Change the appearance of the user interface (page 19).
Settings — For configuring various application settings, including which DDA and DIA protocols will be used by default for data analysis. See Configure application settings (page 19).
Logout — Logs you out of PAS.
Work with PAS tables
Information in PAS is organized into tables. For example, the Projects page (page 83) offers a table of all
projects, while the Users and Permissions page (page 21) offers a table of all PAS users. Use the following techniques when working with tables.
Show or hide table columns
Some tables offer a Show/Hide list from which you can show and hide columns by selecting and clearing checkboxes.
Add custom columns
You can add custom columns to the Plate Samples section (page 78) and Sample List section (page 84). Any custom column you add to one of these tables is available for the other table. You can show and hide custom columns like any other column.
To add a custom column:
1. Select Show/Hide, scroll to the bottom of the list, and select Add Custom to open the Add Custom
Field dialog.
2. Complete the fields.
– Custom Name — A unique name for this custom field, within this table and with no spaces.
– Description — The description of the custom field.
– Notes — Additional information about the custom field.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 12

Chapter 1 Welcome to PAS Tour of the PAS application
3. Select Create.
The custom field (column) appears at the bottom of the Show/Hide column with the prefix custom_.
Sort a table
Sort table items by selecting the heading of the column you want to sort by. PAS sorts in descending order by default. To reverse the order, select the heading again. To turn off sorting entirely, select the heading one more time.
Filter a table; search for a specific item
Filter a table to show only specific items — and to find a specific item — by typing a string of characters in the Search field. As you type, PAS refreshes the table to show only matching items. To show all items again, clear the Search field.
Control how many items are shown per table page
To control how many table items PAS shows per page, select an option from the Items per page list, located at the lower right below the table.
Navigate among table pages
Depending on how many items are shows per page, some tables may be broken into several pages. To navigate among the pages, use the left and right arrows located at the lower left below the table.
Access item details and/or sub-items
Some tables offer access to item details and/or related sub-items. To access an item’s details or its sub-items, select that item’s row. For example, on the Projects page, selecting a project row to access its related samples.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 13

Chapter 1 Welcome to PAS
Tour of the PAS application
Set color preferences in PAS graphs
Many Preferences dialogs for PAS graphs offer controls with which you can set colors to suit your preferences. These controls open a color picker from which you can select a color.
Get help with PAS
To set a color preference:
1. Select the color control, such as Graph Colors as shown here, to open the color picker.
2. Select a color on the color spectrum or enter a color’s hexadecimal value.
3. Click outside color picker to close it.
PAS offers many in-application resources where you can learn how to get the most out of it.
1. On the sidebar menu, select Help to open the Help page.
2. Select the resource you’re interested in: PAS web-based Help, the PDF of the user guide, or training videos.
Manage your user profile
1. On the User Account menu, select User Profile to open the User Profile page.
2. Edit the fields as needed. (Depending on your role and organization, the fields may vary.)
– User Profile — Edit your first name, last name, and phone number.
– Tenant Info — Edit information about your organization and your address.
3. Select Save.
A B
Selected item
Table of sub-items associated with the selected item
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 14

Chapter 1 Welcome to PAS
Input files
Input files
PAS accepts the following files as input for analysis.
• Analysis protocol (.xml, .json) — (Optional) The parameters for an MS database search. PAS includes preinstalled analysis protocols for both data-dependent acquisition (DDA) and data-independent acquisition (DIA) data files.
• MS data files (.raw, .wiff, .wiff.scan) — The results of MS analysis for each sample or control in a plate. Each sample or control is associated with one MS data file.
• Plate map file (.csv) — The location of each sample in a plate with information on each sample, control, nanoparticle, and peptide.
• Sample description file (.csv) — Metadata for each sample in a plate, such as sample name and IDs, type, species, and condition.
Plate map file format
The ProteographTM Instrument Control Software (ICS) generates the plate map file (.csv). All columns are prefilled except tor MS file name, which you must define.
COLUMN DESCRIPTION
MS file name The name of the MS data file, including extension (for example, EXP20101_ 2020ms0321X83_A.raw)
Sample name A unique identifier for the sample, typically the biological sample name1
Sample ID A unique identifier for the sample1
Well location The column and row of the well the sample occupies
Nanoparticle The nanoparticle name (nanoparticle 1–5)
Nanoparticle ID The nanoparticle lot (kit ID)
Control The control type: Process Control, Digestion Control, MPE Control, or MS Peptide Control
Control ID The control lot
Instrument name The name of the liquid handler that converted the samples into peptides
Date sample The date the method ended preparation
Sample volume The volume of sample in the indicated well2
Peptide concentration The concentration of peptide in the well2
Peptide mass sample The mass of peptide in the well2
Recon volume The volume of Peptide Reconstitution Buffer added to the well2
Kit ID The ID of the assay kit used to convert the samples into peptides
Plate ID A unique identifier for the plate
Plate Name A descriptive name for the plate
1The sample name links the plate map and sample description files, while the sample ID groups the nanoparticles in an assay. 2The file shows the numeric value only, without unit of measure.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 15

Chapter 1 Welcome to PAS
Input files
Sample description file format
The sample description file (.csv) contains the following information about each sample in a plate. The file can also include custom (user-defined) columns. These columns must include the prefix custom_.
Table 1. Columns in a sample description file
COLUMN DESCRIPTION
Sample Name A unique identifier for the sample, typically the biological sample name
Sample Type The type of sample, such as plasma or tissue lysate
Species The species (human or mouse) from which the sample was collected
Description A description of the sample
Sample Receipt Date The date your laboratory received the sample
Sample Collection Date The date the sample was collected
Condition The categorical group the sample belongs to
Biological Replicate The biological replicate number
Technical Replicate The technical replicate number
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
16

Chapter 1 Welcome to PAS Output files
Output files
An analysis generates several results files, which record search results for each sample in .txt and .xml formats.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 17

Chapter 1 Welcome to PAS Quality control
Quality control
A plate includes prepared samples and the following controls for quality control (QC) purposes. The control results provide insight into the variation and reproducibility of experiments and LC-MS analysis.
• Process Control — A reference sample added before protein corona formation.
• Digestion Control — A reference sample added before trypsin digestion.
• MPE Control — Reference peptides added before desalting cleanup.
• MS Peptide Control — Reference peptides added before LC-MS analysis.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 18

Chapter 1 Welcome to PAS Application settings
Application settings
You can configure several PAS application settings, including the default analysis protocols for controls and samples. You can also change the appearance of the PAS user interface. These settings are global and apply to all analyses and pages. Finally, you can configure two-factor authentication for yourself.
Change the appearance of the user interface
1. On the User Account menu, select Appearance to open the Appearance page.
2. Select the theme you want to use:
– Light (default) — Makes the background of the user interface white.
– Dark — Makes the background of the user interface black.
3. Select Save.
Configure application settings
Follow these steps to configure PAS application settings. See also Configure two-factor authentication for
yourself (below).
1. On the User Account menu, select Settings to open the Settings page.
2. In the Settings section, configure settings as needed.
– Default Analysis Protocol — Select the DDA-based and DIA-based analysis protocols you want to use for controls and for samples.
– Remove MS data files when plate is deleted — Select either:
. No (default) to have PAS retain the MS data files linked to the plate.
. Yes to have PAS delete the MS data files linked to the plate.
– Receive email notifications on analysis updates — Select either:
. Yes (default) to have PAS send you an email whenever an analysis status changes.
. No to have PAS not send emails about analysis updates.
3. Select Save.
Configure two-factor authentication for yourself
You can configure PAS to require two-factor authentication when you log in. That means that PAS will prompt not only for your username and password, but also for a generated authentication code.
Before continuing, you will need to have already installed an authentication app on your mobile device. PAS is compatible with many such apps, including Google Authenticator, Duo Mobile, 1Password, Auth, and Microsoft Authenticator.
1. On the User Account menu, select Settings to open the Settings page.
2. In the Two-factor Authentication section, select Use Authenticator App to open the Authenticator
verification dialog.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 19

Chapter 1 Welcome to PAS Application settings
3. Using the authenticator app on your mobile device, either scan the QR code or enter the key shown on- screen into the authenticator app.
4. In PAS, enter the generated code.
5. Select Submit and then select OK to close the success message.
Configure default display settings for analysis results
In addition to configuring PAS application settings (see Configure application settings (previous page)), you can also configure display settings for analysis results. These display settings determine which analysis results are
NOTE
Notice that the Use Authenticator App button now reads Disable Authenticator App Security. To turn two-factor authentication off, follow the same steps as above, select that button, and follow the on-screen prompts.
shown
and how they are grouped and sorted. The settings are global and apply to all analyses.
1. On the sidebar menu, select Analyses to open the Analyses page.
2. Select any analysis.
3. Below the Analyses table, locate and select Display Settings to open the Display Settings dialog.
4. Configure settings as needed.
– Group by — Select an option for grouping protein group and peptide counts.
– Sort by — Select an option for sorting data on the Analysis Summary tab.
– — Select or clear checkboxes to show or hide wells. Scroll to see all wells.
– Hide Controls — Set the toggle key to ON to hide controls.
– Visualization graphs — Select each toggle key to ON or OFF to show or hide the corresponding graph.
5. Select Continue to apply the settings.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 20

Chapter 1 Welcome to PAS
User management
User management
If you are a PAS administrator, you can add, edit, and delete users.
Users and Permissions page
If you are an Admin, use the Users and Permissions page to manage PAS users. • To open this page, select Users on the sidebar menu.
Toolbar items
• Add User — Select to add a new user. See Add a user (below).
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item
(page 13). Table columns
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
Name — The user’s name.
Email — The user’s email address.
Role — The user’s role, which grants access to specific PAS functionality.
– User — Can add plates, create projects, create analysis protocols, view MS data files, and view results files.
– Admin — In addition to User functionality, can also manage users. Group — The group or groups the user is assigned to.
Status — The user’s status (e.g., Confirmed, meaning acceptance of the invitation to join PAS). (Edit) — Select to edit the selected user. See Edit a user (next page).
• • •
•
•
• •
1. On the sidebar menu, select Users to open the Users and Permissions page.
2. Select Add User to open the Invite User dialog.
3. Complete the fields. Required fields are marked on-screen with an asterisk.
– Username — Enter a unique identifier for the user.
– Email — Enter the user’s email address.
NOTE
– Use group assignments to limit user access to specific plates, projects, and analyses.
– Members of a user group can access and view the plates, projects, and analyses of everyone in the group.
(Delete) — Select to delete the selected user. See Delete a user (next page). Add a user
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 21

Chapter 1 Welcome to PAS User management
– Role — From the list, select the user’s role: User (the default) or Admin.
– User Groups — (For a user of User role) From the list, select one or more existing user groups. To
add a new group, enter its name.
4. Select Send.
The user will be sent an email invitation with the assigned username, a temporary password, and a link to
PAS.
Edit a user
1. On the sidebar menu, select Users to open the Users and Permissions page.
2. Find the user you want to edit and select (Edit) to open the Edit User dialog.
3. Edit the fields as needed.
4. Select Save.
Delete a user
1. On the sidebar menu, select Users to open the Users and Permissions page.
2. Find the user you want to delete and select (Delete).
3. Select OK to confirm.
NOTE
. Use group assignments to limit user access to specific plates, projects, and analyses.
. Members of a user group can access and view the plates, projects, and analyses of everyone in
the group.
. By default, PAS excludes the user from all groups.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 22

Chapter 2
Analysis Setup
Offers step-by-step guidance for different setup workflows for getting ready for analysis.

Chapter 2 Analysis Setup Analysis setup workflows
Analysis setup workflows
PAS offers several workflows for getting ready for data analysis. To set up a new plate and initiate a new analysis, follow:
• Setup workflow: Add a plate (next page) — This is the most straightforward setup workflow. Begin on the Plates page and add a plate.
• Setup workflow: Link to a plate (page 28) — Begin on the Data Files page, add MS data files, and then link the files to a plate.
To analyze existing plate data, follow:
• Setup workflow: Select samples or controls for analysis (page 31) — Begin on the Projects page to analyze samples. Choose this workflow when you have more than one plate whose samples you want to analyze in a single analysis. You can also choose this workflow when you want to reanalyze samples.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 24

Chapter 2 Analysis Setup Setup workflow: Add a plate
Setup workflow: Add a plate
When you set up analysis by adding a plate, PAS guides you through the workflow with the Add Plate dialog. In this workflow, you add a new plate to PAS and then add samples to a new or existing project. Depending on your choices during the workflow, the analysis will start immediately or will be deferred for later.
To begin this workflow:
1. On the sidebar menu, select Plates to open the Plates page.
2. Select Add Plate to open the Add Plate dialog.
Add one or more MS data files
In the MSData Files section, you add one or more MS data files.
1. Select the MS data files.
– To add a single file, select Files to open the Add Files dialog.
– To add multiple files, select Folder to open the Add Folder dialog.
2. Either drag the file or folder into the drag-and-drop area or use Browse to navigate to and select it.
3. Select Add.
4. Review the selected files.
– To remove a file, select (Delete).
– To remove all files, select Clear.
5. Select Next to advance.
Add the plate map file
In the Plate Map File section, you add a plate map file, which specifies the locations of samples.
1. If you don’t have a plate map file, create one before continuing.
a. Select the on-screen link from which you can download an example plate map file (.csv).
b. Open the file and edit it as needed.
The MS file name, Sample ID, and Plate ID columns are required. (See Plate map file format (page 15) for detailed descriptions of the columns in the file.)
c. Save as a .csv file.
2. Select Add File or Add to open the Add File dialog.
NOTE
Supported file formats are .raw, wiff, or .wiff.scan. Supported folders are RAW and D.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 25

Chapter 2 Analysis Setup Setup workflow: Add a plate
3. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
4. Select Add.
5. Select Next to advance.
Specify plate information
In the Plate ID and Name section, you link the MS data files to a new or existing plate.
1. Do either of the following:
– To link to an existing plate, select Use Existing Plate, and then select a plate from the Select Existing Plate list.
– To set up a new plate, complete the fields.
. Plate ID — A unique identifier for the plate.
. Plate Name — A descriptive name for the plate.
2. Select Next to advance.
Add a sample description file (optional)
In the Sample Description File section, you upload metadata for each sample in the plate.
1. Select Add or Add File to open the Add Files dialog.
2. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
3. Select Add.
4. Select Next to advance.
Add the samples to a project
In the Add to Project section, you add samples to a new or existing project. Depending on your choices, the analysis will start immediately or will be deferred for later.
1. Create or select a project:
– To create a new project, select New Project, enter a project name, and select Add.
– To use an existing project, select it from the Select Project list.
2. Select or clear the applicable checkboxes. (To defer analysis, clear both checkboxes.)
– Analyze samples after addition — Select to analyze samples.
– Analyze controls after addition — Select to analyze controls.
3. Select the MS method:
– DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual
precursor ion.
NOTE
This part of the workflow is optional. To skip it, select Next to advance.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 26

Chapter 2 Analysis Setup Setup workflow: Add a plate
– DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
4. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method), select a protocol.
5. Select Add Plate, and then select Close.
– If you had selected either or both Analyze… checkboxes earlier, the analysis starts immediately.
– If you deferred analysis, you must start it manually. See Start the analysis (below).
Start the analysis
If you deferred analysis during the setup workflow, follow these steps to start the analysis.
1. On the sidebar menu, select Projects to open the Projects page.
2. Select the applicable project.
3. Select one of the following options:
– Analyze — Opens the Analysis dialog from which to analyze all selected samples in the project.
– Analyze Controls — Opens the Analyze Controls dialog from which to analyze the controls only.
4. If you are analyzing all selected samples, in the Analysis Name field, enter a name for the analysis.
5. Select the MS method:
– DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual precursor ion.
– DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
6. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method), select a protocol.
7. In the Description field, enter a description of the analysis.
8. In the Notes field, optionally enter any additional information about the analysis.
9. If you are analyzing all selected samples and want to exclude the controls, select the Exclude controls checkbox.
10. If you are analyzing only controls, under Analysis Name Pattern, review the MS data file name format and list of controls to analyze.
11. Select Start, and then select OK.
The samples or controls are queued for analysis.
12. On the sidebar menu, select Analyses to open the Analyses page.
13. Confirm that the new analysis appears on the Analyses page.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 27

Chapter 2 Analysis Setup Setup workflow: Link to a plate
Setup workflow: Link to a plate
When you set up analysis by linking to a plate, PAS guides you through the workflow with the Link to Plate dialog. In this workflow, you link MS data files and associated sample information to a new or existing plate. Depending on your choices during the workflow, the analysis will start immediately or will be deferred for later.
To begin this workflow:
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Select the folder that holds the files you want to work with.
3. Select the checkbox of each MS data file to include in the analysis. Each MS data file represents one sample.
4. Select Link to Plate to open the Link to Plate dialog.
Review the MS data files to be linked a plate
In the MSData Files section, you review the selected MS data files to be linked to a plate.
1. Review the selected MS data files.
– To remove a file, select its (Delete) button.
– To remove all files, select Clear.
2. Select Next to advance.
Add the plate map file
In the Plate Map File section, you add a plate map file, which specifies the locations of samples.
1. If you don’t have a plate map file, create one before continuing.
a. Select the on-screen link from which you can download an example plate map file (.csv).
b. Open the file and edit it as needed.
The MS file name, Sample ID, and Plate ID columns are required. (See Plate map file format (page 15) for detailed descriptions of the columns in the file.)
c. Save as a .csv file.
2. Select Add File or Add to open the Add File dialog.
3. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
NOTE
If you have not already uploaded the MS data files you want to work with, follow the steps in Upload MS data files (page 90) before starting this workflow.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 28

Chapter 2 Analysis Setup
Setup workflow: Link to a plate
4. Select Add.
5. Select Next to advance.
Specify plate information
In the Plate ID and Name section, you link the MS data files to a new or existing plate.
1. Do either of the following:
– To link to an existing plate, select Use Existing Plate, and then select a plate from the Select Existing Plate list.
– To set up a new plate, complete the fields.
. Plate ID — A unique identifier for the plate.
. Plate Name — A descriptive name for the plate.
2. Select Next to advance.
Add a sample description file (optional)
In the Sample Description File section, you upload metadata for each sample in the plate.
1. Select Add or Add File to open the Add Files dialog.
2. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
3. Select Add.
4. Select Next to advance.
Add the samples to a project
In the Add to Project section, you add the samples to a new or existing project. Depending on your choices, the analysis will start immediately or will be deferred for later.
1. Create or select a project:
– To create a new project, select New Project, enter a project name, and select Add.
– To use an existing project, select it from the Select Project list.
2. Select or clear the applicable checkboxes. (To defer analysis, clear both checkboxes.)
– Analyze samples after addition — Select to analyze samples.
– Analyze controls after addition — Select to analyze controls.
3. Select the MS method:
– DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual
precursor ion.
– DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
NOTE
This part of the workflow is optional. To skip it, select Next to advance.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 29

Chapter 2 Analysis Setup Setup workflow: Link to a plate
4. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method), select a protocol.
5. Select Add Plate, and then select Close.
– If you had selected either or both Analyze… checkboxes earlier, the analysis starts immediately.
– If you deferred analysis, you must start it manually. See Start the analysis (below).
Start the analysis
If you deferred analysis during the setup workflow, follow these steps to start the analysis.
1. On the sidebar menu, select Projects to open the Projects page.
2. Select the applicable project.
3. Select one of the following options:
– Analyze — Opens the Analysis dialog from which to analyze all selected samples in the project.
– Analyze Controls — Opens the Analyze Controls dialog from which to analyze the controls only.
4. If you are analyzing all selected samples, in the Analysis Name field, enter a name for the analysis.
5. Select the MS method:
– DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual precursor ion.
– DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
6. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method), select a protocol.
7. In the Description field, enter a description of the analysis.
8. In the Notes field, optionally enter any additional information about the analysis.
9. If you are analyzing all selected samples and want to exclude the controls, select the Exclude controls checkbox.
10. If you are analyzing only controls, under Analysis Name Pattern, review the MS data file name format and list of controls to analyze.
11. Select Start, and then select OK.
The samples or controls are queued for analysis.
12. On the sidebar menu, select Analyses to open the Analyses page.
13. Confirm that the new analysis appears on the Analyses page.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 30

Chapter 2 Analysis Setup Setup workflow: Select samples or controls for analysis
Setup workflow: Select samples or controls for analysis
The Projects page is the starting point for the sample selection method of analysis setup.
Choose this workflow when you have more than one plate whose samples you want to analyze in a single
analysis. You can also use this workflow to reanalyze samples. See Analyze selected samples (below). Use this workflow also to analyze controls only. See Analyze controls only (next page).
NOTE
For this workflow, PAS must already contain the applicable plate and MS data files in a project.
Figure 1. Projects page (left) and Analysis dialog (right)
Analyze selected samples
1. On the sidebar menu, select Projects to open the Projects page.
2. Select the applicable project.
3. Select the checkbox of each sample to analyze.
4. Select Analyze to open the Analysis dialog.
5. In the Analysis Name field, enter a name for the analysis.
6. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method),
select a protocol.
7. In the Description field, enter a description of the analysis.
8. In the Notes field, optionally enter any additional information about the analysis.
9. If you want to exclude the controls, select the Exclude controls checkbox.
10. Select Start, and then select OK. The samples are queued for analysis.
11. On the sidebar menu, select Analyses to open the Analyses page.
12. Confirm that the new analysis appears on the Analyses page.
A Selected samples
B Analyze button
C Analysis dialog
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 31

Chapter 2 Analysis Setup Setup workflow: Select samples or controls for analysis
Analyze controls only
1. On the sidebar menu, select Projects to open the Projects page.
2. Select the applicable project.
3. Select samples, and then select Analyze Controls to open the Analyze Controls dialog.
4. Select the MS method:
– DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual precursor ion.
– DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
5. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method), select a protocol.
6. In the Description field, enter a description of the analysis.
7. In the Notes field, enter any additional information about the analysis.
8. Under Analysis Name Pattern, review the MS data file name format and the list of controls to analyze.
9. Select Start, and then select OK.
The samples are queued for analysis.
10. On the sidebar menu, select Analyses to open the Analyses page.
11. Confirm that the new analysis is listed.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 32

Chapter 3
Analysis Results
Describes analysis results (exclusive of Group Analysis) and offers step-by-step guidance for working with them.

Chapter 3 Analysis Results Access analysis results
Access analysis results
Access analysis results on the Analyses page, where you can review data in a variety of graphs and download results files. The results of the selected analysis are arranged in up to five tabs, each presenting a subset: Analysis Summary, Analysis Metrics, Group Analysis, QC Charts, and Analysis Output Files.
NOTE
Depending on the selected analysis, the QC Charts tab might not be shown.
Figure 2. Analyses page
A Select an analysis C
B Select tabs to view subsets of the analysis’s
results D
Download the analysis’s protein groups and peptide results
A results graph
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
34

Chapter 3 Analysis Results Review analysis results
Review analysis results
1. On the sidebar menu, select Analyses to open the Analyses page.
2. From the Status list, select Completed.
3. To apply additional filters, select options from any of the following lists:
– Project — Shows analyses for specified projects.
– Plate — Shows analyses for specified plates.
– Controls/Samples — Shows analyses for controls only or samples only.
4. Select an analysis.
5. On the Analysis Summary tab, compare samples and review a summary of the analysis, counts of peptides and protein groups, the dynamic range distribution of detected proteins, sample overlap, and sample correlation. To learn more about this tab, begin at Analysis Summary tab (page 37).
6. On the Analysis Metrics tab, review the intensities, sample clusters, nanoparticle counts, and other QC metrics. To learn more about this tab, begin at Analysis Metrics tab (page 44).
7. If the analysis included controls, select the QC Charts tab to review the control results. To learn more about this tab, begin at QC Charts tab (page 53).
8. On the Analysis Output Files tab, review results files.
9. (Optional) Use Protein Group / Peptide Results to download the analysis’s protein groups and
peptide results. See Download an analysis’s protein groups and peptides results (page 87).
NOTE
If you select multiple analyses, PAS does not show an analysis summary or results files and, instead, groups all data in the graphs on the remaining tabs.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 35

Chapter 3 Analysis Results Data visualization
Data visualization
Several tabs of the Analyses page (page 86) are divided into expandable and collapsible sections. Some sections include a menu for downloading graph data as a .svc, .png, or .csv file. Sections with many graphs include scroll arrows for accessing all the graphs.
Figure 3. Example section
A Scroll arrows and page numbers to move through all graphs
B Arrow to expand or collapse the section
C Menu for downloading data in the section
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 36

Chapter 3 Analysis Results Analysis Summary tab
Analysis Summary tab
An analysis’s Analysis Summary tab (on the Analyses page) offers an overall view of analysis results, organized into sections.
For some sections, you can modify labels, reorganize layouts, and otherwise adjust display preferences. These modifications are temporary and apply only to the select analysis. When you leave the Analyses page, each section returns to the default view. (To configure display preferences for all analyses in PAS, see Configure default display settings for analysis results (page 20).)
Open an analysis’s Analysis Summary tab
1. On the sidebar menu, select Analyses to open the Analyses page.
2. From the Status list, select Completed.
3. Select an analysis to open its analysis results.
4. Select the Analysis Summary tab.
Summary section
The Summary section shows the following information.
• Analysis name — The name of the analysis.
• Number of samples — The number of samples analyzed.
• Peptides across — The total number of distinct peptides across samples.
• Run types — The analysis protocol type, either DDA or DIA.
• Protein group across — The total number of distinct protein groups across samples.
• Peptide average — The average number of peptides per sample.
• Analysis protocol — The name of the analysis protocol applied to the analysis.
• Protein group average — The average number of protein groups per sample.
Protein Group Counts section
The Protein Group Counts section graphs the number of proteins in each sample. The y-axis plots the number of identified protein groups and the x-axis plots each well. Three lines compare protein group trends for the plate:
• The individual line traces the total number of protein groups observed in each sample.
• The cumulative line traces the cumulative number of protein groups observed with each successive sample. For example, the A3 value reflects the number of protein groups detected at least one time among A1, A2, and A3.
• The common line traces the proteins in common for each successive sample. For example, the A3 value reflects the number of proteins in common with A1, then A2.
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 37

Chapter 3 Analysis Results Analysis Summary tab
Dots along each line identify values for each well.
• Hover over a dot on the line graph to view the exact value.
• Select a dot in the legend to hide the corresponding line.
• Hover over a dot in the legend to hide the lines corresponding to the other dots.
To customize this section for the selected analysis, see Set Protein Group Counts preferences (below). Figure 4. Line graph showing protein group counts
Set Protein Group Counts preferences
You can customize the appearance of the Protein Group Counts section (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Summary tab. (See Open an analysis’s Analysis Summary tab (previous page).)
2. In the Protein Group Counts section, select (Settings) to open the Preferences dialog.
3. Set preferences as needed.
– X Tick Label — Select the label to show along the x-axis, e.g., Sample Name, Plate Name, Sample ID.
– Y Axis Range — Select the range for the y-axis.
. Min is 0 and Max Data Dependent — The minimum value is set at 0 and the maximum value depends on data.
. Min and Max Data Dependent — Both the minimum and maximum values depend on data.
. User Defined — Enter the minimum and maximum values.
4. Select Continue to apply the settings.
Peptide Counts section
The Peptide Counts section graphs the number of peptides in each sample. The y-axis plots the peptide count, and the x-axis plots each sample. You can update the x-axis label, but the values remain the same. Hover over a dot on the line graph to view the exact value.
To customize this section for the selected analysis, see Set Peptide Counts preferences (next page).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 38

Chapter 3 Analysis Results Analysis Summary tab
Figure 5. Line graph showing peptide counts
Set Peptide Counts preferences
You can customize the appearance of the Peptide Counts section (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Summary tab. (See Open an analysis’s Analysis Summary tab (page 37).)
2. In the Peptide section, select (Settings) to open the Preferences dialog.
3. Adjust the settings as you prefer.
– X Tick Label — Select the label to show along the x-axis, e.g., Sample Name, Plate Name, Sample ID.
– Y Axis Range — Select the range for the y-axis.
. Min is 0 and Max Data Dependent — The minimum value is set at 0 and the maximum value depends on data.
. Min and Max Data Dependent — Both the minimum and maximum values depend on data.
. User Defined — Enter the minimum and maximum values.
4. Select Continue to apply the settings.
Distribution of Detected Proteins in Plasma Proteome section
The Distribution of Detected Proteins in Plasma Proteome section shows the dynamic range of proteins identified in each sample compared to a deep reported human plasma proteome index.1 The n-value equals the number of proteins detected in the sample that were also detected in the index.
The x-axis plots the number of identified proteins ranked by database intensity. The y-axis plots the intensity calculated in the database using the logarithm base 10 (log10) function. Each protein appears as a circle on the plot.
• Hover over a circle to view the name of the protein and the exact intensity values.
• Use the scroll arrows and numbers to move through all the graphs.
• Select or clear wells in the Samples list to show or hide the corresponding graphs.
• Enter a keyword or term in the Search field to find specific graphs.
• Annotate proteins you want to highlight, as shown in red in the following figure.
To customize this section for the selected analysis, see Set Distribution of Detected Proteins in Plasma
Proteome preferences (next page).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 39

Chapter 3 Analysis Results Analysis Summary tab
Figure 6. Graph comparing the proteins detected in sample A3 to the plasma proteome
1Keshishian, Hasmik, Michael W. Burgess, Michael A. Gillette, Philipp Mertins, Karl R. Clauser, D. R. Mani, Eric W. Kuhn, et al., “Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury,’ 14, no. 9 Molecular & Cellular Proteomics (September 2015): 2375–2393, https://doi.org/10.1074/mcp.M114.046813.
Set Distribution of Detected Proteins in Plasma Proteome preferences
You can customize the appearance of the Distribution of Detected Proteins in Plasma Proteome section (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Summary tab. (See Open an analysis’s Analysis Summary tab (page 37).)
2. In the Distribution of Detected Proteins in Plasma Proteome section, select (Settings) to open the
Preferences dialog.
3. Adjust the settings as you prefer.
– Graph Colors — Use the color picker to select graph colors. (See Set color preferences in PAS graphs (page 14).)
– Page Chart Layout — Select an option for how many graphs appear on a page and the layout. For example, the 2 × 2 option shows four graphs per page, two down and two across.
– Quantiles — Adjust the frequency distribution. The default quantiles are 0.25, 0.5, and 0.75.
. To add a quantile, select Add and enter its value.
. To modify existing quantiles, update the values in each field.
. To delete a quantile, select (Delete).
– Proteins — Annotate proteins of interest by entering their names, separated by commas (e.g., P35579,F5H7Y6,Q5VZ73). The dots representing the annotated proteins will appear on each graph in red with the protein names above.
4. Select Continue to apply the settings.
Protein Group Overlap Sets section
The Protein Group Overlap Sets section is divided into two bar graphs and a matrix that together show protein group intersections. The Within Samples checkbox shows intersection size and protein group count as standalone graphs for each sample with a dot representing the MS data file.
• Hover over a vertical bar to view the intersection size for the group of samples as an integer and the percentage of the total it represents.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 40

Chapter 3 Analysis Results Analysis Summary tab
• Hover over a horizontal bar to view the sample and the exact number of protein groups it contains.
• Select or clear wells in the Samples list to show or hide the corresponding bars.
To customize this section for the selected analysis, see Set Protein Group Overlap Sets preferences (below). Figure 7. Graphs and a matrix show protein group overlaps
A Intersection Size bar graph C Matrix
B Protein Group Count bar graph
Set Protein Group Overlap Sets preferences
You can customize the appearance of the Protein Group Overlap Sets section (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Summary tab. (See Open an analysis’s Analysis Summary tab (page 37).)
2. In the Protein Group Overlap Sets section, select (Settings) to open the Preferences dialog.
3. Adjust the settings as you prefer.
– Horizontal Bar Position — Select an orientation for the graph.
. Left — The graph is positioned left.
. Right — The graph is positioned right.
. None — The graph is hidden.
– Vertical Height Rate — Enter a value for the relative bar height in the graph.
– MS file’s name length — Enter the character limit for the MS data file name.
– Display Top N Intersection — Select which bars appear on the graph.
. All — Shows intersection sizes for all samples.
. 4 — Shows the intersection sizes of the top four largest samples only.
. 8 — Shows the intersection sizes of the top eight largest samples only.
. 16 — Shows the intersection sizes of the top 16 largest samples only.
. 32 — Shows the intersection sizes of the top 32 largest samples only.
– Display Percentage — Select to show percentages on top of each bar in the Intersection Size graph.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 41

Chapter 3 Analysis Results Analysis Summary tab
– Page Chart Layout — Select an option for how many graphs appear on a page and the layout. For example, the 2 × 2 option shows four graphs per page, two down and two across.
By default, this section consolidates information for all samples into two bar graphs and a matrix for easy comparison. You can divide these elements into graphs for individual samples and change the page layout accordingly.
– — Set colors for the various parts of the graph, such as for the vertical and horizontal bars. Use the techniques in Set color preferences in PAS graphs (page 14).
4. Select Continue to apply the settings. Sample Comparability section
The Sample Comparability section shows the degree of statistical correlation between samples based on the Pearson correlation coefficient (PCC) and the similarity in protein groups between samples based on the Jaccard Index, which measures the linear correlation of data. You can switch between the PCC and Jaccard index.
Each matrix is color-coded for easy reference. Samples on the green end of the spectrum have high correlation. Samples on the red end of the spectrum have low correlation.
To customize this section for the selected analysis, see Set Sample Comparability preferences (below). Figure 8. A color-coded matrix shows sample comparability data using PCC (left) or the Jaccard index (right)
Set Sample Comparability preferences
You can temporarily customize the appearance of the Sample Comparability section (above). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Summary tab. (See Open an analysis’s Analysis Summary tab (page 37).)
2. In the Sample Compatibility section, select (Settings) to open the Preferences dialog.
3. Adjust the settings as you prefer.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 42

Chapter 3 Analysis Results Analysis Summary tab
– Similarity Score — To change how sample intensity data are compared, select either:
. Pearson’s correlation coefficient — Shows data calculated by normalizing the covariance
measurement.
. Jaccard index — Shows data calculated by dividing the intersection size by the union size.
– Color Labels By — To change the matrix label colors, select an option, e.g., Sample Name, Sample ID, Plate Name. To not use colors for labels, select Disabled.
– Label Font Size — Select the point size text of the label.
4. Select Continue to apply the settings.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 43

Chapter 3 Analysis Results Analysis Metrics tab
Analysis Metrics tab
An analysis’s Analysis Metrics tab (on the Analyses page) shows the QC metrics for an analysis result, organized into sections. The metrics indicate how well an analysis method performed.
For some sections, you can modify labels, reorganize layouts, and otherwise adjust display preferences. These modifications are temporary and apply only to the select analysis. When you leave the Analyses page, each section returns to the default view. (To configure display preferences for all analyses in PAS, see Configure default display settings for analysis results (page 20).)
Open an analysis’s Analysis Metrics tab
1. On the sidebar menu, select Analyses to open the Analyses page.
2. From the Status list, select Completed.
3. Select an analysis to open its analysis results.
4. Select the Analysis Metrics tab.
Intensities section
The Intensities section graphs the protein and peptide intensities and the distribution of protein sequence coverage for each sample, including the coefficient of variation (CV). You can update the default chart type, a density plot, to a violin plot.
On a density plot, the x-axis plots intensity and the y-axis plots density.
• Move the pointer along a curve to view the exact intensity value at each point in the curve.
• Select a sample in the legend to hide the corresponding line.
• Hover over a sample in the legend to hide the lines corresponding to the other samples.
On a violin plot, the sample appears along the x-axis and the y-axis plots intensity. Box plots represent quantile ranges with box plots with density on either end. Red dots represent outliers.
• Hover over a box plot to view the quantile for the sample.
• Hover over a dot to view the exact outlier value.
To customize this section for the selected analysis, see Set Intensities preferences (next page).
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 44

Chapter 3 Analysis Results Analysis Metrics tab
Figure 9. Density plot (left) and violin plot (right) showing the intensity distribution for a sample
Set Intensities preferences
You can temporarily customize the appearance of the Intensities section (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Metrics tab. (See Open an analysis’s Analysis Metrics tab (previous page).)
2. In the Intensities section, select (Settings) to open the Preferences dialog.
3. Adjust the settings as you prefer.
– Page Chart Layout — Select an option for how many graphs appear on a page and the layout. For example, the 2 × 2 option shows four graphs per page, two down and two across.
– Chart Type — Select an option.
. Density plot — Shows the full distribution of data for each sample as a continuous curve.
. Violin plot — Shows data for each sample as quartiles with the full distribution of data.
4. Select Continue to apply the settings.
Plate Map Grid section
The Plate Map Grid section shows metrics for each sample in the format of 96-well plates. Each plate represents one of the following metrics. Hover over a well to view the value. Alternatively, you can consolidate these data into a single table with one column for each metric.
• Protein group counts — The number of protein groups identified per well.
• Peptide counts — The number of peptides identified per well.
• Peptide quant mass — The mass of peptide in the well as calculated by the peptide quantification assay.
• Miscleavage rate — The fraction of peptides identified as having missed cleavages based on the maximum allowable missed cleavages set in the analysis protocol.
• Oxidation ratio — The fraction of peptides identified with methionine oxidation.
• ID rate — The rate at which an MS/MS scan is converted into a database peptide identification.
To customize this section for the selected analysis, see Set Plate Map Grid preferences (next page).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 45

Chapter 3 Analysis Results Analysis Metrics tab
Figure 10. Grids for each metric showing results for each sample
Set Plate Map Grid preferences
You can temporarily customize the appearance of the Plate Map Grid section (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Metrics tab. (See Open an analysis’s Analysis Metrics tab (page 44).)
2. In the Plate Map Grid section, select (Settings) to open the Preferences dialog.
3. Adjust the settings as you prefer.
– Graph Types — Select each toggle key to show or hide a graph or table column (depending on the Display Type).
– Scaling — Select Each graph or Each type scaling for reviewing multiple plates.
– Display Type — Select an option:
. Plate Map Grid — Shows each metric in a separate grid.
. Table — Shows the protein group counts, peptide counts, and other metrics in one table.
– Page Chart Layout — Select an option for how many graphs appear on a page and the layout. For
example, the 2 × 2 option shows four graphs per page, two down and two across.
4. Select Continue to apply the settings.
Lamppost Proteins’ Concentration section
The Lamppost Proteins’ Concentration section graphs the intensity of landmark proteins in each sample. The x-axis plots each well and plate and the y-axis plots log10 intensity.
Five lines compare intensities for the proteins HP, APOC3, PROS1, F11, and ALB. Dots along each line identify the intensity value for each protein in the sample.
• Hover over a dot on the line graph to view the exact value for all five proteins.
• Select a dot in the legend to hide the corresponding line.
• Hover over a dot on the legend to hide the lines corresponding to the other dots.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 46

Chapter 3 Analysis Results Analysis Metrics tab
To customize this section for the selected analysis, see Set Lamppost Proteins’ Concentration preferences (below). Specifically, you can add, edit, and delete proteins.
Figure 11. Line graph showing intensities for landmark proteins
Set Lamppost Proteins’ Concentration preferences
You can temporarily customize the appearance of the Lamppost Proteins’ Concentration section (previous page). Specifically, you can add, edit, and delete proteins to use instead of the default QC landmark proteins. The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Metrics tab. (See Open an analysis’s Analysis Metrics tab (page 44).)
2. In the Lamppost Proteins Concentration section, select (Settings) to open the Lamppost Proteins
dialog.
3. To add a protein or group of proteins:
a. Select Add Proteins to open the Add Proteins dialog.
b. In the Name field, enter a label for a detected protein or group of proteins.
c. In the Proteins field, enter a protein name or enter multiple names separated by commas. d. Select Add.
Notice that the label you entered for the protein or group of proteins is now selected.
4. To edit a protein or group of proteins:
TIP
To revert to using the default QC landmark proteins, select Abundance monitoring.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 47

Chapter 3 Analysis Results Analysis Metrics tab
a. Select its (Edit) button to open the Edit Proteins dialog.
b. Change the fields as needed.
c. Select Edit to save the changes.
5. To delete a protein or group of proteins:
a. Select its (Delete) button.
b. Select OK to confirm.
6. Select Continue to apply the settings.
PCA Analysis section
The PCA Analysis section clusters similar samples in a scatter plot. The x-axis plots principal component 1 (PC1) analysis scores and the y-axis plots principal component 2 (PC2) analysis scores. Each dot represents one sample.
• Select a dot under the x-axis to hide the corresponding sample.
• Hover over a dot to view the sample and plate names.
To customize this section for the selected analysis, see Change the PCA Analysis color scheme (below). Figure 12. Scatter plot showing clusters of similar samples
Change the PCA Analysis color scheme
You can temporarily change the color scheme of the PCA Analysis section (above). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Metrics tab. (See Open an analysis’s Analysis Metrics tab (page 44).)
2. In the PCA Analysis section, select an option from the Color By list:
– Condition — Applies the same color to all data points. The color represents sample quality.
– Sample Name — Applies a unique color to each sample.
– Plate Name — Applies the same color to all data points. The color represents the sample plate.
– Sample ID — Applies a unique color to each sample.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 48

Chapter 3 Analysis Results Analysis Metrics tab
3. Set colors as you prefer.
a. Select a label box next to or below the Color By list. For example, if you selected Sample ID
above, you can adjust the color for each sample.
b. Use the color picker to select a color. (See Set color preferences in PAS graphs (page 14).)
c. Repeat for other label boxes.
Peptide Counts Distribution and Protein Group Counts Distribution sections
The Peptide Counts Distribution and Protein Group Counts Distribution sections show the peptide and protein counts for the five nanoparticles as box plots. A table below each plot shows the analysis name, mean value, and CV percentage for each nanoparticle. Each box plot and table represent one plate.
• Hover over a dot to view the peptide or protein count, file, and sample name.
• Hover over a box to view the quantile for the nanoparticle.
Figure 13. Box plots showing peptide counts distribution
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 49

Chapter 3 Analysis Results Analysis Metrics tab
Figure 14. Box plots showing protein group counts distribution
Protein Group Counts of Nanoparticles and Peptide Counts of Nanoparticles sections
The Protein Group Counts of Nanoparticles and Peptide Counts of Nanoparticles sections offer two charts:
• Protein Group Counts — Shows the sample protein group counts identified by each nanoparticle. Each
dot represents a sample. The x-axis plots samples and the y-axis plots the number of peptides.
• Peptide Counts — Shows the sample peptide counts identified by each nanoparticle. Each dot represents a sample. The x-axis plots samples and the y-axis plots the number of peptides.
To use these charts:
• Hover over a dot to view the well, nanoparticle that occupies the well, and the peptide count.
• Select a dot under the x-axis to remove the corresponding well from the plot.
To customize this section for the selected analysis, see Set Protein Group Counts of Nanoparticles and Peptide
Counts of Nanoparticles preferences (next page).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 50

Chapter 3 Analysis Results Analysis Metrics tab
Figure 15. Scatter plot showing protein group counts of nanoparticles
Figure 16. Scatter plot showing peptide counts of nanoparticles
Set Protein Group Counts of Nanoparticles and Peptide Counts of Nanoparticles preferences
You can temporarily customize the appearance of the Protein Group Counts of Nanoparticles and Peptide Counts of Nanoparticles sections (previous page). The next time you view the section, it will have reverted to its default view.
1. Open the Analysis Metrics tab. (See Open an analysis’s Analysis Metrics tab (page 44).)
2. In the Protein Group Counts of Nanoparticles or Peptide Counts of Nanoparticles section, select
(Settings) to open the Preferences dialog.
3. Adjust the settings as you prefer.
– Y Axis Range — Select the range for the y-axis.
. Min is 0 and Max Data Dependent — The minimum value is set at 0 and the maximum value
depends on data.
. Min and Max Data Dependent — Both the minimum and maximum values depend on data.
. User Defined — Enter the minimum and maximum values.
4. Select Save.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 51

Chapter 3 Analysis Results Analysis Metrics tab
Hierarchical Clustering section
The Hierarchical Clustering section shows a cluster analysis based on agglomerative nesting, which groups samples in clusters based on similarity. Each sample starts a single cluster. Pairs of clusters are then successively merged until all clusters are merged into a dendrogram.
Figure 17. Dendrogram successively grouping similar samples
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 52

Chapter 3 Analysis Results QC Charts tab
QC Charts tab
An analysis’s QC Charts tab (on the Analyses page) shows assay control data for an analysis. Each chart plots one metric for one assay control category. Charts are organized in a grid, with the same metric across each row and the same control type down each column. The x-axis is labeled with the date of analysis and the y-axis depends on the metric.
• Select a status icon (e.g., ) in the summary table above the control charts to jump to the corresponding control chart.
• Hover over a dot on a control chart to view general information about the control, such as well and plate.
Toolbar items
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
•
• •
• • •
• •
•
— By default, all controls are included. Select another option to filter by a specific control type.
– Process Control — Results for the process controls only.
– Digestion Control — Results for the digestion controls only.
– MPE Control — Results for the MPE controls only.
– MS Peptide Control — Results for the mass spec controls only.
MS Instrument — The instruments listed vary depending on your organization. Select up to six instruments to aggregate their data.
Color By — (Available when only one MS instrumented is selected.) Select your preferred coloration for the graphs, e.g., Gradient.
Start Date — Enter or select the earliest date of data you want to see. End Date — Enter or select the latest date of data you want to see Control Limits — Select how you want control limits to be defined.
– –
–
Preinstalled — Use the preinstalled control limits.
User Defined — Define your own control limits. See Define and use user-defined control limits
(page 56).
Calculated — Calculate control limits from data within one or more date ranges, which you define with the Calculate button (see below).
Limits — Select to view the calculated control limit date ranges. From here, you can also delete a range. See Delete a calculated control limit date range (page 56).
Exclusion — Select to exclude an outlier or other data point from the control limit calculation. Selecting one data point (sample) in a plot excludes the corresponding sample from the other plots. A well containing the mass spec control has 10 reported QC metrics. All other control wells have 11 reported QC metrics. See Exclude a sample (page 56).
Calculate — Select to define a calculated control limit based on a range of dates. See Define and use calculated control limits (page 56).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 53

Chapter 3 Analysis Results QC Charts tab
• Annotate — Select to annotate all control charts for a specific date. See Annotate control charts (page 57).
• Reports — Select to generate a PDF report of control results, with specific criteria. See Generate a detailed PDF report of control results (page 58).
Figure 18. Results for controls
A Filters for viewing charts for all controls or a selected control type
B Toolbar with additional filters and functions
Open and filter an analysis’s QC Charts tab
C Summary of control data for the selected analysis time frame
D QC charts with metrics of each control
1. On the sidebar menu, select Analyses to open the Analyses page.
2. From the Status list, select Completed.
3. From the Controls/Samples list, select Samples included.
4. Select the checkbox of each applicable analysis whose QC charts you want to work with.
5. Select the QC Charts tab.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 54

Chapter 3 Analysis Results
QC Charts tab
The QC Charts tab (page 53) presents each control with metrics for assay and MS performance.
6. Under MS Instrument, select up to six instruments.
The tab aggregates data from the selected instruments and hides the Color By menu.
7. To filter the results for a specific control type, select an option:
– Process Control — Results for the process controls only.
– Digestion Control — Results for the digestion controls only.
– MPE Control — Results for the MPE controls only.
– MS Peptide Control — Results for the mass spec controls only.
8. To filter results for a specific time frame, enter the MS injection time frame in the Start Date and End Date fields.
Set QC Charts preferences
1. Open and filter the QC charts tab for an analysis. (See Open and filter an analysis’s QC Charts tab
(previous page).)
2. Select (Settings) to open the Preferences dialog.
3. Complete the fields as needed.
– Show Annotations — Select to show or hide annotation. See Annotate control charts (page 57).
– Show Historical Data — Select to show or hide historical data. By default, each control chart shows values from historical data. Although independent of the selected analyses, these data points are sourced from previous analyses that exist in the user group.
– Show Variables — Select the checkbox of each variable of interest, e.g., Protein Group Count, Peak Width (seconds). Clear the checkbox of any you’re not interested in.
– Process Variables Y Axis Range — Select the range for the y-axis for processed variables.
. Min and Max Data Dependent — Both the minimum and maximum values depend on data.
. Min is 0 and Max Data Dependent — The minimum value is set at 0 and the maximum value depends on data.
. User Defined — Enter the minimum and maximum values.
4. Select Save.
Manage user-defined and calculated control limits
Depending on the MS injection time frame, filtering results on the QC Charts tab by time frame shows a summary that presents each control with metrics for assay and MS performance. Each metric is color-coded to indicate whether it is within the control limits (teal) or outside the control limits (orange).
Control limits help determine whether results are expected. The mean provides a historical average, while the upper and lower control limits indicate normal variation. PAS includes default control limits calculated using two standard deviations above and below the mean. Calculated control limits are generated from user data.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 55

Chapter 3 Analysis Results QC Charts tab
Define and use user-defined control limits
1. On the Controls section’s toolbar, select the Control Limits list and select User Defined to open the Control Limits dialog.
2. Along the top of the dialog, select the tab of the control whose limits you want to define, e.g., MPE Control. Use the scroll arrows to view all tabs.
3. For each control limit, enter a value.
– Mean — The value to serve as the historical mean.
– UCL — The value to serve as the upper control limit.
– LCL — The value to serve as the lower control limit.
4. Select Save.
Define and use calculated control limits
1. On the Controls section’s toolbar, select Calculate to open the Calculate Control Limits dialog.
2. In the Start Date and End Date fields, enter or select the applicable dates.
3. Select Save.
A teal background appears on the plots, indicating that data are shown with modified control limits.
4. On the toolbar, select the Control Limits list and select Calculated to use the calculated control limits.
Delete a calculated control limit date range
1. On the Controls section’s toolbar, select Limits to open the Calculate Control Limits Range dialog.
2. Find the date range you want to delete and select its (Delete) button.
3. Select Yes to confirm.
4. Select Save.
Exclude a sample
Exclude an outlier or other data point from the control limit calculation. Selecting one data point (sample) in a plot excludes the corresponding sample from the other plots. A well containing the mass spec control has 10 reported QC metrics. All other control wells have 11 reported QC metrics.
1. Select Exclusion to open the Control Limit Exclusion dialog and display a list of samples that are being excluded.
2. Find the data point (sample) you want to remove and select its (Delete) button.
3. Select Yes to confirm.
4. Select Close.
NOTE
The new control limit date range has been added to the Calculate Control Limits Range dialog, which you can open with the Limits button.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
56

Chapter 3 Analysis Results QC Charts tab
Annotate control charts
You can add an annotation that will be applied to all control charts for a specific date. An annotation is indicated by a dot above a control chart with the full annotation below the chart. Select an annotation dot to view its annotation type, date, and accompanying message.
Add an annotation
1. Open and filter the QC charts tab for an analysis. (See Open and filter an analysis’s QC Charts tab (page 54).)
2. Select Annotate to open the Annotate dialog.
3. Complete the fields.
– Annotation Type — Do either of the following:
. To select an existing annotation type, select it from the list.
. To add a new annotation type, select Add Annotation Type, enter the annotation, and select Add.
– Date — Enter or select the date the event occurred.
– Message — Enter more information about the annotation.
4. Select Save.
Edit an annotation
1. Select the annotation dot for the annotation you want to edit.
2. In the annotation pop-up, select (Edit) to open the Annotate dialog.
3. Edit the annotation as needed.
4. Select Save.
Delete an annotation
1. Select the annotation dot for the annotation you want to delete.
2. In the annotation pop-up, select (Delete).
3. Select OK to confirm.
Generate reports of control results
Summarize and output control results in a .csv file or generate a more detailed report as a .pdf file. The summary lists each QC metric with the control, well, plate, value, and result. The report summarizes results and shows a table for each control. Each table includes the well and plate and lists values for each QC metric with optional notes.
Download summarized control results
1. Open and filter the QC charts tab for an analysis. (See Open and filter an analysis’s QC Charts tab (page 54).)
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 57

Chapter 3 Analysis Results QC Charts tab
2. In the summary table above the control charts, select (Download CSV) to open the Download CSV dialog.
3. Select the checkbox of each field to add to the summary:
– File name — The full name of the MS data file, including extension.
– Assay instrument name — The name of the liquid handler that prepared the assay.
– Mass spec instrument name — The name of the MS instrument that analyzed the assay output.
– Injection time — The month and day of MS injection.
– Gradient — The duration of the LC gradient.
4. Select a format:
– One line per metric — Organizes the summary by metric.
– One line per control — Organizes the summary by control.
5. Select Download to generate the summary.
Generate a detailed PDF report of control results
1. Open and filter the QC charts tab for an analysis. (See Open and filter an analysis’s QC Charts tab (page 54).)
2. Select Reports to open the Controls Report dialog.
3. Complete the fields.
– Assay Equipment ID — Select the name of the liquid handler used to prepare the assay.
– Mass Spec ID — Select the name of the MS instrument that analyzed the assay output.
– Report By — (Read-only) Shows your username.
– Report Date — (Read-only) Shows the current date and time.
4. Select Create to generate the report.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 58

Chapter 3 Analysis Results Analysis Output Files tab
Analysis Output Files tab
An analysis’s Analysis Output Files tab (on the Analyses page) lists all outputs of the search engine results for the analysis. You can download an output file simply by selecting it.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 59

Chapter 4
Analysis Interpretation
Describes techniques for interpreting analysis results, specifically for group analyses, performed from the Group Analysis tab of the Analyses page.

Chapter 4 Analysis Interpretation
Open an analysis’s Group Analysis tab
Open an analysis’s Group Analysis tab
1. On the sidebar menu, select Analyses to open the Analyses page.
2. From the Status list, select Completed.
3. Select an analysis to open its analysis results.
4. Select the Group Analysis tab. (See Group Analysis tab (page 66).)
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
61

Chapter 4 Analysis Interpretation Export raw data to a file
Export raw data to a file
Prior to doing a group analysis, you can export raw data to a .csv file.
1. Open the Group Analysis tab. (See Open an analysis’s Group Analysis tab (previous page).)
2. Select Export.
3. Navigate to the location on your computer where you want to save the file and select Save.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 62

Chapter 4 Analysis Interpretation View a raw protein group data heatmap
View a raw protein group data heatmap
Prior to doing a group analysis, you can visualize a heatmap of raw protein group intensity.
1. Open the Group Analysis tab. (See Open an analysis’s Group Analysis tab (page 61).) 2. Select Heatmap.
PAS prepares the data and then displays the heatmap.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 63

Chapter 4 Analysis Interpretation Set up and start a group analysis
Set up and start a group analysis
Follow these steps to prepare to conduct a group analysis in which you visualize differences in proteins detected between groups.
To begin this workflow:
1. Open the Group Analysis tab. (See Open an analysis’s Group Analysis tab (page 61).)
2. Select Setup Group Analysis to open the Setup Group Analysis dialog.
Continue to the next section.
Specify the quantitation level and grouping of samples
In the Feature and Grouping section, you specify the parameters for the differential expression analysis between groups of samples.
1. For Feature, select the quantitation level, either Protein or Peptide.
2. For Group By, select the category by which you want to group samples, e.g., by a condition or custom
field. The options vary based on the metadata imported from the sample description file.
3. Select Next to advance.
Filter out (exclude) samples and groups
In the Filter Samples and Group section, you specify the individual samples or whole groups of samples to be filtered out of (excluded from) the final comparison.
1. (Optional) From Select Sample, select one or more samples.
– If you change your mind about an excluded sample, select its (Delete) button.
2. (Optional) From Select Groups, select one or more groups of samples.
– If you change your mind about an excluded group, select its (Delete) button.
3. Select Next to advance.
Filter out (exclude) proteins or peptides
In the Filter Peptides / Proteins section, you specify measurement completeness thresholds for the final comparison.
1. Complete the fields.
NOTE
PAS will use whichever minimum (percentage or specific value) yields the larger number of samples.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 64

Chapter 4 Analysis Interpretation Set up and start a group analysis
– Minimum % of valid values in at least one group — Enter the minimum percentage of samples to consider. For example, if there are 24 samples and you enter 50, a minimum of 12 samples will be considered. The default is 0.
– Minimum number of valid values in at least one group — Enter the exact minimum number of samples to consider. The default is 2.
– Remove peptides / proteins with complete missing values in at least one group — Select to exclude peptides / proteins for which data is entirely missing (i.e., all table cells that show -).
– Remove peptides / proteins with missing values — Select to exclude peptides / proteins with one or more missing values (i.e., some table cells that show -).
2. Select Next to advance.
Normalize values and impute sparse or missing values
In the Normalization and Imputation section, you specify the processes by which raw measurement values are normalized and sparse and missing values are handled.
1. For Normalization, select either:
– Median — Raw MS intensity values are normalized on a run-by-run basis. This will account for
potential measurement bias and make samples more comparable.
– None — No normalization of raw measurements will occur.
2. For Imputation, select either:
– Minimal Probability — Substitute missing values with random values from a normal distribution using a mean that is down-shifted from the sample mean and a standard deviation (SD) that is a fraction of the SD of the sample distribution.
– None — No imputation of raw measurements will occur.
3. Select Next to advance.
Select the statistical test and the groups to compare
In the Statistical Test and Group Comparison section, you select the statistical test to use for comparison analysis and then select the groups of samples you want to compare.
1. For Statistical Test, select the appropriate test to use for comparison analysis.
– T Test — A parametric test. Select this if you previously elected to normalize data.
– Wilcoxon — A non-parametric text rank-based test. Select this if you previously elected not to normalize data.
2. Under Groups for Comparison, select the checkbox of each set of groups you want to compare. The groups offered vary, based on selections you made earlier in the setup workflow.
3. Select Start.
The group analysis begins immediately. When it is finished, the analysis results are shown on the Group Analysis tab, organized into sections, with the Volcano section being shown. To explore all the sections, begin at Group Analysis tab (next page).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 65

Chapter 4 Analysis Interpretation Group Analysis tab
Group Analysis tab
An analysis’s Group Analysis tab (on the Analyses page) shows the analysis and results from comparing two user-defined groups, organized into sections. You can visualize differences in proteins detected between the groups and explore initial biological insights into the data.
Toolbar items
• Before you run a group analysis, the toolbar includes:
–
– – –
– –
Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item (page 13).
Feature — Select Protein or Peptide to filter the Raw Data table.
Group By — Select the categorical grouping by which you want to group raw data.
Heatmap — (Not applicable to peptides.) Select to visualize a heatmap of raw protein group data. See View a raw protein group data heatmap (page 63)
Export — Select to export raw data to a .csv file. See Export raw data to a file (page 62).
Setup Group Analysis — Select to set up groups for and start a comparative analysis. See Set
up and start a group analysis (page 64).
• After you run a group analysis, the toolbar includes these additional items:
–
– – –
– –
–
–
–
Raw Data / Results — Use to switch back and forth between the raw data and group analysis results.
Significant Proteins — Select to show only significant proteins.
Volcano Plot — Select to view the Volcano section (page 69).
Coverage — (Available when at least two proteins are selected.) Select to view the Coverage section (page 70).
Clustered Heatmap — Select to view the Clustered Heatmap section (page 68).
PPI Network — (Available only for significant proteins.) — Select to view the Filtered PPI
Network section (page 71).
Enrichment — (Available only for significant proteins.) — Select to view the Enrichment section
(page 72).
Box Plots — (Available only for significant proteins.) — Select to view the Box Plots section
(page 73).
Preferences — Select to set threshold preferences. e.g., High Regulation Threshold.
Raw Data section
The Raw Data section shows a table consisting of protein groups (rows) identified across the analyzed samples (columns) and their corresponding intensity levels (values). Samples for which a protein group was detected show a numerical value. However, samples for which a protein group was not detected show a hyphen – character.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 66

Chapter 4 Analysis Interpretation Group Analysis tab
• Search for the protein (protein group) to extract intensity values across samples.
• Select the protein to visualize the distribution of abundances between samples corresponding to each
group as an Intensity Plot box plot.
• Select the Heatmap button to visualize a heatmap of raw protein group data.
Figure 19. Raw Data table listing proteins and Intensity Plot for the selected protein
Figure 20. Raw data table listing proteins and heatmap for all proteins
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 67

Chapter 4 Analysis Interpretation Group Analysis tab
Clustered Heatmap section
After doing a group analysis, you can view the Clustered Heatmap section.
Here you can visualize protein abundances across samples, with both proteins (rows) and samples (columns)
clustered based on agglomerative nesting. Cell colors range from blue (lowly abundant) to red (highly abundant).
• Use the + and – buttons to zoom in and out of the graph.
• Hover over a cell to reveal the protein name, sample name, and abundance.
• Select (“hamburger menu”) to download the graph as .svg or .png.
Figure 21. Clustered heatmap graph
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 68

Chapter 4 Analysis Interpretation Group Analysis tab
Volcano section
After doing a group analysis, you can view the Volcano section.
Here you can visualize a scatter plot showing the statistical significance (p-value) in abundance differences between your compared groups versus the magnitude of the change (Fold Change). Highlighted in the teal quadrants are proteins with large fold changes (default: ±2) and statistically significant (default: p < 0.05). Data displayed in the plot are shown in detail in the table on the left of the plot.
• Each point in the plot represents a protein. Hover over each point to display the protein group, fold change, and p-value.
• Clear the Significant Proteins checkbox in the Group Analysis section’s toolbar to display both significant and non-significant proteins.
• Select this section’s (Settings) button to highlight a protein of interest.
• Select (“hamburger menu”) to download the graph as .svg or .png.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 69

Chapter 4 Analysis Interpretation Group Analysis tab
Figure 22. Volcano graph
Coverage section
After doing a group analysis, you can view the Coverage section.
Here you can visualize the amino acid sequence of your selected protein and the regions where measured
peptides map. You must select at least one protein in the list to the left of this section. Coverage is represented as percent (%) of sequence observed by measured peptides.
• In cases where a PTM is detected for your selected protein, use the Coverage list to change to PTM to view where the PTM occurs.
• Select (Expanded View) to expand the section so you can see data more easily, without its wrapping
• Select (“hamburger menu”) to download the graph as .svg or .png.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 70

Chapter 4 Analysis Interpretation Group Analysis tab
Figure 23. Coverage graph
Filtered PPI Network section
After doing a group analysis and with the Significant Proteins checkbox selected, you can view the Filtered PPI Network section.
Here you can visualize a network plot showing protein-protein interactions between proteins with significantly different abundances between groups compared.
• Select this section’s (Settings) button to adjust the confidence of the Minimum Interaction Score.
• Nodes in the graph are selectable to view protein description and can be moved around.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 71

Chapter 4 Analysis Interpretation Group Analysis tab
Figure 24. Filtered PPI Network graph
Enrichment section
After doing a group analysis and with the Significant Proteins checkbox selected, you can view the Enrichment section.
Here you can functionally characterize proteins showing abundance difference between groups compared by performing gene ontology (GO) enrichment.
• Use the list to switch between GO categories: Molecular Function, Biological Process, and Cellular Compartment.
• Select this section’s Chart Settings button to set the cutoff (adjusted p-value upper limit).
• Display the GO term enrichment as either:
– Dot Plot — To “condense” similar terms by family (i.e., condense all children from each superfamily), select Summarize Terms.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 72

Chapter 4 Analysis Interpretation
Group Analysis tab
–
.
. .
Ontology Plot (hierarchical tree)
Hover over a box to view its GO information.
Use the mouse wheel to zoom into and out of the plot.
Drag the plot to reposition it so that you can examine different areas.
Figure 25. Enrichment plot, shown as an ontology plot (hierarchical tree)
Box Plots section
After doing a group analysis and with the Significant Proteins checkbox selected, you can view the Box Plots section.
Here you can visualize the intensity differences between groups of significantly different proteins.
• Select this section’s (Settings) button to set layout preferences for the charts.
• Hover over a dot to view a sample’s count and name.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 73

Chapter 4 Analysis Interpretation Group Analysis tab
Figure 26. Box plots
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 74

Chapter 5
Data Management
Covers the components of and how to work with PAS’s main pages.

Chapter 5 Data Management Plates and samples
Plates and samples
A plate in PAS represents a Peptide Collection Plate, which is the output of the Proteograph Assay method analyzed with MS. A plate contains the samples, controls, and nanoparticles from a method run with the corresponding metadata.
Plates page
Use the Plates page to manage plates and sample information and to upload custom files. • To open this page, select Plates on the sidebar menu.
Toolbar items
• Add Plate — Select to add a plate to PAS when setting up an analysis. See Setup workflow: Add a plate (page 25).
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item (page 13).
• Show/Hide — Select or clear checkboxes to show or hide table columns. See Show or hide table columns (page 12).
Table columns
• Name — The name of the plate.
• Plate ID — The unique identifier of the plate.
• Description — The description of the plate.
• Notes — Additional information about the plate.
• Created By — The user who created the plate.
• Created Time — The date and time the plate was created.
• Last Modified By — The user who last modified the plate.
• Last Modified Time — The date and time the plate was last modified.
• ID — The unique, internal identifier of the plate.
• (Add Sample) — Select to add samples to the selected plate. See Add samples to an existing plate (page 79).
• (Edit) — Select to edit the selected plate. See Edit a plate (next page). Other page sections
• Plate Samples — Shows a table of samples associated with the plate selected in the Plates section. For detailed information about this section, see Plate Samples section (page 78).
• Plate Grid — Shows a grid or table of wells associated with one or more samples selected in the Plate Samples section. For detailed information about this section, see Plate Grid section (page 81).
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 76

Chapter 5 Data Management Plates and samples
Review a plate
1. On the sidebar menu, select Plates to open the Plates page.
2. Select a plate to review.
The Plate Samples table appears, listing the samples in the selected plate.
3. (Optional) Adjust the table view as needed:
– To adjust the number of visible table rows, scroll to the bottom of the table and select the number of items per page.
– To sort a table column, select the column heading to sort in descending order. To sort in ascending order, reselect the column.
– To filter samples, enter a keyword or term in the Search field.
4. Select samples for review:
– To review all samples, select the checkbox in the column heading.
– To review selected samples, select each sample’s checkbox.
The Plate Grid appears, showing the plate with yellow wells indicating the selected samples.
5. Review sample details:
– To individually review sample details, select a yellow well.
– To simultaneously review details for all samples, select Table View.
– To modify sample details, see Edit or delete samples in wells (page 81).
Edit a plate
1. On the sidebar menu, select Plates to open the Plates page.
2. Find the plate you want to edit and select its (Edit) button to open the Edit Plate dialog.
3. Edit the fields as needed.
– Plate ID — Enter a unique identifier for the plate.
– Plate Name — Enter a descriptive name for the plate.
– Description — (Optional) Enter a description of the plate.
– Notes — (Optional) Enter additional information about the plate.
4. Select Confirm to save.
Delete a plate
1. On the sidebar menu, select Plates to open the Plates page.
2. Find the plate you want to delete and select its (Delete) button.
3. Select OK to confirm.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 77

Chapter 5 Data Management Plates and samples
Plate Samples section
The Plate Samples section is part of the Plates page (page 76).
• To open it, select Plates on the sidebar menu and then select a plate. You many need to scroll down to see the Plates Samples section.
Toolbar items
• Link to Sample Description File — Select to link a sample description file to the selected plate. See Add sample descriptions (page 80).
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item (page 13).
• Show/Hide — Select or clear checkboxes to show or hide table columns. See Show or hide table columns (page 12).
Table columns
• Name — The descriptive name of a sample.
• Sample/Control ID — The unique identifier of a sample or control.
• Well Location — The location of a sample or control in the selected plate.
• Type — The type of sample: Plasma or Peptide.
• Species — The species a sample was collected from: Human or Mouse.
• Sample Collection Date —The date the sample was collected.
• Sample Receipt Date — The date your laboratory received the sample.
• Condition — The categorical group the sample belongs to.
• Biological Replicate — The biological replicate number.
• Technical Replicate — The technical replicate number.
• Description — The description of the sample.
• Notes — Additional information about the sample.
• Created By — The user who created the sample.
• Created Time — The date and time of the sample was created.
• Last Modified By — The user who last modified the sample.
• Last Modified Time — The date and time the sample was last modified.
• ID — The unique, internal identifier of the sample.
• Plate ID — The unique identifier of the plate.
• Plate Name — The descriptive name of the plate.
• (Edit) — Select to edit information about the selected sample. See Edit sample information (page 80).
• (Delete) — Select to delete the selected sample. See Delete a sample from a plate (page 81).
TIP
You can add custom columns to this table. See Add custom columns (page 12).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 78

Chapter 5 Data Management Plates and samples
Add samples to an existing plate
Adding samples to an existing plate follows a similar workflow to the add-a-plate workflow of analysis setup (described in Setup workflow: Add a plate (page 25)). The difference is that in the add-a-plate workflow, you add a new plate and its samples to PAS, whereas you can also add samples to an existing plate, as described below.
1. On the sidebar menu, select Plates to open the Plates page.
2. Find the plate to which you want to add sample and select its (Add Sample) button to open the Add
Sample dialog.
3. In the MSDATA Files section, you add one or more MS data files.
a. Select the MS data files.
– To add a single file, select Files to open the Add Files dialog.
– To add multiple files, select Folder to open the Add Folder dialog.
b. Either drag the file or folder into the drag-and-drop area or use Browse to navigate to and select
it.
c. Select Add.
d. Review the selected files.
– To remove a file, select (Delete).
– To remove all files, select Clear.
e. Select Next to advance.
4. In the Plate Map File section, you add a plate map file, which specifies the locations of samples.
a. If you don’t have a plate map file, create one before continuing.
A. Select the on-screen link from which you can download an example plate map file (.csv).
B. Open the file and edit it as needed.
The MS file name, Sample ID, and Plate ID columns are required. (See Plate map file format (page 15) for detailed descriptions of the columns in the file.)
C. Save as a .csv file.
b. Select Add File or Add to open the Add File dialog.
c. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
d. Select Add.
e. Select Next to advance.
5. In the Plate ID and Name section, you link the MS data files to an existing plate.
a. Select Use Existing Plate, and then select the applicable plate.
b. Select Next to advance.
6. (Optional) In the Sample Description File section, you upload metadata for each sample in the plate.
NOTE
Supported file formats are .raw, wiff, or .wiff.scan. Supported folders are RAW and D.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 79

Chapter 5 Data Management Plates and samples
NOTE
This part of the workflow is optional. To skip it, select Next to advance.
a. Select Add or Add File to open the Add Files dialog.
b. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
c. Select Add.
d. Select Next to advance.
7. In the Add to Project section, you add the samples to a new or existing project.
a. Create or select a project:
– To create a new project, select New Project, enter a project name, and select Add.
– To use an existing project, select it from the Select Project list.
b. Select or clear the applicable checkboxes. (To defer analysis, clear both checkboxes.)
– Analyze samples after addition — Select to analyze samples.
– Analyze controls after addition — Select to analyze controls.
c. Select the MS method:
– DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual precursor ion.
– DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
d. From the Analysis Protocol list (which shows only protocols compatible with the selected MS method), select a protocol.
e. Select Add Plate, and then select Close.
– If you had selected either or both Analyze… checkboxes earlier, the analysis starts
immediately.
– If you deferred analysis, you must start it manually. See Start the analysis (page 27).
Add sample descriptions
1. On the sidebar menu, select Plates to open the Plates page.
2. In the Plates table, select a plate to add sample descriptions to.
3. On the Plate Samples section’s toolbar, select Link to Sample Descriptions File to open the Upload File dialog.
4. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
5. Select Upload.
The information from the uploaded file appears in the Plate Samples section of the Plates page.
Edit sample information
1. On the sidebar menu, select Plates to open the Plates page.
2. Select the plate for which you want to edit information about a sample.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 80

Chapter 5 Data Management
Plates and samples
3. In the Plate Samples table, find the sample you want to edit and select its Edit window.
4. Complete the fields as needed.
5. Select Confirm to save.
Delete a sample from a plate
(Edit) button to open the
1. On the sidebar menu, select Plates to open the Plates page.
2. Select the plate from which you want to delete a sample.
3. In the Plate Samples table, find the sample you want to delete and select its (Delete) button.
4. Select OK to confirm.
Plate Grid section
The Plate Grid section of the Plates page shows the selected plate with yellow wells indicating the samples selected in the Plate Samples list.
The section offers two views of the same information. Grid View facilitates editing and deleting samples one well at a time, while Table View offers a more efficient means to work with samples in multiple wells. For detailed instructions, see Edit or delete samples in wells (below).
If you have created a custom .csv, .tsv, .xls, or .xlsx file containing sample information, you can upload it to the Plates page from the Plate Grid section. See Upload a custom file containing sample information (next page).
Edit or delete samples in wells
The Plate Grid section of the Plates page offers two views for editing samples in and deleting samples from wells. Grid View facilitates editing and deleting samples one well at a time, while Table View offers a more efficient means to work with samples in multiple wells.
1. On the sidebar menu, select Plates to open the Plates page.
2. Select the plate whose samples you want to edit or delete.
3. In the Plate Samples table, select the checkbox of each sample you want to work with.
The Plate Grid appears below the Plate Samples table and shows the selected samples as yellow wells in a 96-well plate.
4. Edit or delete samples as described below.
Grid view
1. Select Grid View if it is not already selected.
2. To edit a sample in a well:
a. Select a yellow well to open its details and then select its (Edit) button.
b. Edit values in the editable fields. PAS saves each change as you work.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 81

Chapter 5 Data Management Plates and samples
c. When finished editing, select the (Edit) button again to save all the changes. 3. To delete a sample from a well :
a. b.
Table view
Select a yellow well to open its details and then select its (Delete) button. Select OK to confirm.
Table View.
– To edit an individual cell, enter a value, and then press Enter.
1. Select
2. To edit samples in a well:
– To apply the same value to all cells in a column, select the column heading’s enter a value, and then select Apply.
(Update) button,
– To copy and paste a row, scroll all the way to the right and select (Copy). Edit the copied row as
needed and then select (Save Copy).
3. To delete a sample from a well, select the well’s (Delete) button. Select OK to confirm.
Upload a custom file containing sample information
You can create a custom .csv, .tsv, .xls, or .xlsx file containing sample information and upload it from the Plates page. To create the file, match the columns in the file to the columns in the table view.
1. On the sidebar menu, select Plates to open the Plates page.
2. Select the applicable plate.
3. In the Plate Samples table, select the checkbox of at least one sample to open the Plate Grid sections.
4. Select (Upload File) to open the Upload File dialog.
5. Either drag the file into the drag-and-drop area or use Browse to navigate to and select it.
6. Select Upload.
PAS populates the plate grid with imported sample information.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 82

Chapter 5 Data Management Projects
Projects
When you set up an analysis, PAS creates a corresponding project and adds it to the Projects page. The project lists all samples in an analysis with sample information. On the Projects page, you can add samples to the analysis and then start the analysis.
Projects page
Use the Projects page to manage projects, to manage the samples associated with a project, and to view the MS data files associated with a specific sample.
• To open this page, select Projects on the sidebar menu.
Toolbar items
• Add Project — Select to add a new project. See Add a project (next page).
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item
(page 13).
• Show/Hide — Select or clear checkboxes to show or hide table columns. See Show or hide table
columns (page 12).
Table columns
• Tenant ID — The user identification.
• Name — The name of the project.
• Description — The description of the project.
• Notes — Additional information about the project.
• Created By — The user who created the project.
• Created Date — The date and time of project was created.
• Last Modified By — The user who last modified the project.
• Last Modified Date — The date and time the project was last modified.
• ID — The unique, internal identifier of the project.
• (Edit) — Select to edit the selected project’s name, description, and/or notes.
• (Delete) — Select to delete the selected project (one that you originally added). See Delete a project
(next page). You cannot delete someone else’s project (unless you are an Admin). Other page sections
• Sample List — Shows a table of samples associated with the project selected in the Projects section. For detailed information about this section, see Sample List section (next page).
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
TIP
You can add custom columns to this table. See Add custom columns (page 12).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 83

Chapter 5 Data Management Projects
• MS Data files — Shows a read-only table of MS data files associated with the sample selected in the Samples List section. You can show or hide columns and can search for a specific data file.
Add a project
1. On the sidebar menu, select Projects to open the Projects page.
2. Select Add Project to open the Create Project dialog.
3. Complete the fields.
– Project Name — Enter a unique name for the project.
– Select Plate — Select a plate to add to the project.
– Add — Select to add another plate to the project. Repeat as needed.
– Description — Enter a description of the project.
– Notes — Enter any additional information about the project.
4. Select Save.
Delete a project
You can delete any project that you originally created. You cannot delete someone else’s project unless you are an administrator.
1. On the sidebar menu, select Projects to open the Projects page.
2. Find the project you want to delete and select its (Delete) button.
3. Select Yes to confirm.
Sample List section
The Sample List section of the Projects page (previous page) shows a table of samples associated with the
selected project.
• To open it, select Projects on the sidebar menu and then select the project whose samples you want to manage.
Toolbar items
• Add Sample — Select to add samples to the project. See Add samples to a project (next page).
• Analyze — Select to select samples for analysis, as described in Setup workflow: Select samples or
controls for analysis (page 31).
• Analyze Controls — Select to select controls only for analysis. See Analyze controls only (page 32).
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item (page 13).
• Show/Hide — Select or clear checkboxes to show or hide table columns. See Show or hide table columns (page 12).
TIP
You can add custom columns to this table. See Add custom columns (page 12).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
84

Chapter 5 Data Management Projects
Table columns
• Tenant ID — The user identification.
• Name — The descriptive name of a sample.
• Sample/Control ID — The unique identifier of a sample or control.
• Well Location — The location of a sample or control in the selected plate.
• Type — The type of sample: Plasma or Peptide.
• Species — The species a sample was collected from: Human or Mouse.
• Sample Collection Date —The date the sample was collected.
• Sample Receipt Date — The date your laboratory received the sample.
• Condition — The categorical group the sample belongs to.
• Biological Replicate — The biological replicate number.
• Technical Replicate — The technical replicate number.
• Plate Name — The descriptive name of the plate.
• Description — The description of the sample.
• Notes — Additional information about the sample.
• Created By — The user who created the sample.
• Created Date — The date and time of the sample was created.
• Last Modified By — The user who last modified the sample.
• Last Modified Date — The date and time a sample was last modified.
• ID — The unique, internal identifier of the sample.
Add samples to a project
1. On the sidebar menu, select Projects to open the Projects page.
2. Select a project to add samples to.
3. Above the Sample List table, select Add Sample to open the Add Sample dialog.
4. Complete the fields.
– Select Plate — Select the plate to add samples to.
– Select Sample — Select a sample to add.
– Add — Select to add another sample.
5. Select Save to add the selected samples.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 85

Chapter 5 Data Management Analyses
Analyses
When you set up an analysis, PAS adds the analysis to the Analyses page with general information about the analysis: the number of MS data files in the analysis, who last modified the analysis and when, and optional notes and descriptions. An icon to the left of the analysis name indicates the status. Selecting an icon opens the analysis log.
Analyses page
Use the Analyses page to manage analyses and to view and download analysis results and logs. • To open this page, select Analyses on the sidebar menu.
Toolbar items
• Project — Select the project whose analysis data you want to work with.
• Plate — Select the plate whose analysis data you want to work with.
• Controls/Samples — Select whether to show controls or samples on the list.
• Status — Select the status of analyses that you’re interested in, e.g., Completed.
• Refresh — Select to refresh the table.
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item
(page 13).
• (Menu) — Select to open a menu of options (e.g., Delete) to use when multiple analyses are selected.
See Delete one or more analyses (page 88).
• Show/Hide — Select or clear checkboxes to show or hide table columns. See Show or hide table
columns (page 12).
Table columns
• — The status of an analysis: (Succeeded), (Failed).
• Name — The name of an analysis.
• Description — The description of the analysis.
• Notes — Additional information about the analysis.
• Protocol — The protocol used for the analysis.
• MS Data Files — The number of MS data files associated with the analysis.
• Analyzed By — The user who created the analysis.
• Analysis Start — The date and time the analysis started.
• Analysis End — The date and time the analysis ended.
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
TIP
You can add custom columns to this table. See Add custom columns (page 12).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 86

Chapter 5 Data Management
Analyses
• • •
• •
Last Modified By — The user who last modified the analysis.
Last Modified Time — The date and time the analysis was last modified. ID — The unique, internal identifier of the analysis.
(Edit) — Select to edit the selected analysis. See Edit an analysis (below).
(Delete) — Select to delete the selected analysis. See Delete one or more analyses (next page).
Download an analysis log
1. On the sidebar menu, select Analyses to open the Analyses page.
2. Find the analysis whose log you want to download and select the status icon (e.g., (Succeeded)) that
precedes its name.
The Analysis Log dialog opens, listing all analysis events from oldest to newest.
3. Select (Download Log).
4. Depending on your browser or browser preferences, the file may download immediately, or you may be
prompted where to save the file. Follow any on-screen prompts.
Download an analysis’s protein groups and peptides results
You can download results for protein groups and peptides for a selected analysis. The results are organized into several files, each offering simplified data tables containing summarized results.
• Peptide_NP.tsv — Peptides, abundances, and other metadata across nanoparticles.
• Peptide_Panel.tsv — Peptides, abundances, and other metadata with nanoparticle values rolled up.
• Protein_Group_NP.tsv — Protein groups, abundances, and other metadata across nanoparticles.
• Protein_Group_Panel.tsv — Protein groups, abundances, and other metadata with nanoparticle values rolled up.
To download analysis results:
1. On the sidebar menu, select Analyses to open the Analyses page.
2. Select the analysis whose results you want to download.
3. Select Protein Group / Peptide Results, located to the right of the of the analysis’s tabs.
4. Depending on your browser or browser preferences, the file may download immediately, or you may be prompted where to save the file. Follow any on-screen prompts.
Edit an analysis
1. On the sidebar menu, select Analyses to open the Analyses page.
2. Find the analysis you want to edit and select its (Edit) button to open the Edit Analysis dialog.
3. Edit the fields as needed.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 87

Chapter 5 Data Management Analyses
– Analysis Name — Enter a name of the analysis.
– Description — (Optional) Enter a description of the analysis.
– Notes — (Optional) Enter additional information about the analysis.
4. Select Save.
Delete one or more analyses
To delete a single analysis:
1. On the sidebar menu, select Analyses to open the Analyses page.
2. Find the analysis you want to delete and select its (Delete) button.
3. Select OK to confirm.
To delete multiple analyses:
1. On the sidebar menu, select Analyses to open the Analyses page.
2. Select the checkbox of each analysis you want to delete.
3. Select (Menu) and then select Delete.
4. Select OK to confirm.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 88

Chapter 5 Data Management MS data files
MS data files
When you upload MS data files during analysis setup, PAS creates a folder to contain the files. From the Data Files page, you can customize any folder and manually create folders.
Use the following filters to refine your view of the Data Files page.
• Start Date and End Date — Filter for folders and files created within a specified date range.
• Last Uploaded — Filter for folders and files uploaded within the last day, last five days, or last month. Data Files page
Use the Data Files page to create, move, and delete MS data files and folders and to download an application for automatic file updates.
• To open this page, select Data Files on the sidebar menu.
Toolbar items
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
•
• •
• • • •
• •
Link to Plate — (Available when one or more files or folders are selected.) Select to link the selected files or folders to a plate when setting up an analysis. See Setup workflow: Link to a plate (page 28).
New — Select to add a new, empty folder.
Upload — Select to upload data files. See Upload MS data files (next page).
Refresh — Select to refresh the table.
Start Date — Set the earliest creation date for the files you want to view.
End Date — Set the latest creation date for the files you want to view.
Last Uploaded — Select a filter the list to show only those files created or uploaded within the last day (1D), last five days (5D), or last month (1M). To clear the active filter, select it again.
(Menu) — Select to move, rename, or delete the selected files or folders. See Move MS data files (next page), Rename MS data files or folders (next page), and Delete MS data files or folders (page 91).
Uploader — Select to download the Seer AutoUploader application with which you can automatically transfer new MS data files to PAS. You may find it a more convenient means of uploading a large set of
files rather than using the Upload button. See Download and install the Seer AutoUploader application (page 91).
Table columns
• <folders/files> — Initially shows the top level of folders into which data files are organized. Select a folder to view its contents. The column’s heading shows the breadcrumb (path) to the folder whose contents are listed. To return to another folder in the path, select its link in the breadcrumb. To return to
the top-level folders, select (Home).
• Filepath — Shows the location where a file or folder is stored.
• Date — Shows the date the file or folder was added to PAS.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 89

Chapter 5 Data Management MS data files
• •
• •
Time — Shows the time the file or folder was added to PAS. Size — Shows the disk size of the file or folder.
(Download) — Select to download the selected file.
(Delete) — Select to delete the selected file or folder. See Delete MS data files or folders (next page).
Create a folder
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Select New to open the Create Folder dialog.
3. Enter the name of the new folder.
4. Select Create.
Upload MS data files
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Select Upload to open the Add Raw MSDATA Files dialog.
3. Select the MS data files.
– To upload a single file, select Add Files to open the Add Files dialog.
– To upload multiple files, select Add Folder to open the Add Folder dialog.
4. Either drag the file or folder into the drag-and-drop area or use Browse to navigate to and select it.
5. Select Upload.
6. Review the uploaded files.
– To remove a file, select (Delete).
– To remove all files, select Clear.
Move MS data files
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Select the folder where the MS data files you want to move are stored.
3. Select the checkbox of each file you want to move.
4. Select (Menu) and then select Move.
5. Select a destination folder, and then select Move Here.
6. Select OK to confirm.
Rename MS data files or folders
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Navigate to the folder where the folder or file you want to rename is stored.
3. Select the checkbox of the folder or file you want to rename.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 90

Chapter 5 Data Management
MS data files
4. Select (Menu) and then select Rename.
5. Enter a new name for the folder or file.
6. Select Save, and then select OK.
Delete MS data files or folders
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Navigate to the folder where the folder or file you want to delete is stored.
3. Find the folder or file you want to delete and select its (Delete) button.
4. Select OK to confirm.
Download and install the Seer AutoUploader application
The Seer AutoUploader is an application that runs on your MS computer to automatically upload new MS data files to your PAS account. You may find it a more convenient means of uploading a large set of files rather than
using the Upload button on the Data Files page.
1. On the sidebar menu, select Data Files to open the Data Files page.
2. Select
Uploader to open the Download AutoUploader dialog.
a. From Select file to download, select the version of the AutoUploader you want to download.
b. In the Save As window, navigate to the folder in which you want to save the installation package
and select Save.
c. Select Done.
3. In your computer’s file system, navigate to and double-click the installation package to launch it. Follow the prompts to install the software.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 91

Chapter 5 Data Management
Analysis protocols
Analysis protocols
An analysis protocol specifies search parameters for the MS database search engine.
• For DDA-based analyses, PAS uses the MaxQuant1 protocol.
• For DIA-based analyses, PAS uses either the EncyclopeDIA2 or DIA-NN3 protocol.
PAS includes several preinstalled analysis protocols, identified on the Protocols page (below) by the icon. You can also create custom protocols by copying an existing protocol or by uploading a protocol.
(star)
1Cox, Jürgen, and Matthias Mann, “MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification,” 26 Nature Biotechnology (November 2008): 1367–1372, https://doi.org/10.1038/nbt.1511.
2Searle, Brian C., Lindsay K. Pino, Jarrett D. Egertson, Ying S. Ting, Robert T. Lawrence, Brendan X. MacLean, Judit Villén, et al., “Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry,” 9 Nature Communications (December 2018): 5218, https://doi.org/10.1038/s41467-018-07454- w.
3Demichev, V., Messner, C.B., Vernardis, S.I. et al., “DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput.” 17 Nat Methods (January 2020): 41–44, https://doi.org/10.1038/s41592- 019-0638-x.
Protocols page
Use the Protocols page to manage analysis protocols and to view analysis parameters. • To open this page, select Analysis Protocol on the sidebar menu.
Toolbar items
TIP
Use (Collapse) and (Expand) (where available) to selectively collapse and expand sections as you work.
• Copy — (Shown when a protocol is selected.) Select to copy a custom (user-defined) or preinstalled protocol. See Copy an analysis protocol (page 94).
• Upload — Select to upload a protocol. See Upload an analysis protocol (next page).
• Search — Use to filter the table or find a specific item. See Filter a table; search for a specific item
(page 13).
• Show/Hide — Select or clear checkboxes to show or hide table columns. See Show or hide table
columns (page 12).
Table columns
• Name — The name of the protocol.
• Type — Whether the associated MS method is DDA or DIA.
TIP
You can add custom columns to this table. See Add custom columns (page 12).
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 92

Chapter 5 Data Management Analysis protocols
• Species — Whether the analyzed sample is from a human or mouse.
• Version — The version of the protocol, useful if you want to keep track of different versions (updates) of
protocols.
• Description — The description of the protocol.
• Notes — Additional information about the protocol.
• Created By — The user who created the protocol.
• Created Time — The date and time the protocol was created.
• Parameters — The name of the file in which the search engine parameters are defined. To view the search engine parameters for a protocol, select its row. See View an analysis protocol’s search engine parameters (below).
• Preinstalled — Shows which protocols are preinstalled (indicated by (star)). You may copy but not delete preinstalled protocols.
• ID — The unique, internal identifier of the protocol.
• (Download) — Select to download a protocol. See Download an analysis protocol (next page).
• (Delete) — Select to delete selected protocol. See Delete an analysis protocol (next page). View an analysis protocol’s search engine parameters
1. On the sidebar menu, select Analysis Protocol to open the Protocols page.
2. Select the analysis protocol whose search engine parameters you want to view.
A panel opens to the right of the Protocols table to display the selected protocol’s parameters, which are read in from the protocol’s parameters file.
Upload an analysis protocol
1. On the sidebar menu, select Analysis Protocol to open the Protocols page.
2. Select Upload to open the Analysis Protocol dialog.
3. Complete the fields.
– Name — Enter a unique name for the protocol.
– Analysis Type— Select an MS method.
From the DDA list, select the MS method:
. DDA — Derives an MS/MS spectra from selection, isolation, and fragmentation of an individual
precursor ion.
. DIA — Derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
– Analysis Engine — Select an analysis engine, e.g., Max Quant.
For information about the analysis engines supported by PAS, see Analysis protocols (previous
page).
– Species — Select the species to analyze: Human or Mouse.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 93

Chapter 5 Data Management
Analysis protocols
– Description — Enter a description for the protocol.
– Notes — Enter any additional information about the protocol.
4. When ready to upload the protocol:
a. Select Upload to open the Upload File dialog.
b. Drag-and-drop the file or select Browse to navigate to and select the file.
c. Select Upload.
5. On the Analysis Protocol dialog, select Save.
Copy an analysis protocol
1. On the sidebar menu, select Analysis Protocol to open the Protocols page.
2. Select the protocol you want to copy and select Copy.
– For a DDA-based protocol, be sure to copy a DDA protocol.
– For a DIA-based protocol, be sure to copy a DIA protocol. The DDA and DIA fields cannot be modified in a copied protocol.
3. In the Edit Protocol dialog, edit fields for the new protocol.
4. Select Save.
Download an analysis protocol
1. On the sidebar menu, select Analysis Protocol to open the Protocols page.
2. Find the protocol you want to download and select its (Download) button.
3. On the Save As dialog, navigate to the folder where you want to save the protocol and select Save.
Delete an analysis protocol
1. On the sidebar menu, select Analysis Protocol to open the Protocols page.
2. Find the custom protocol you want to delete and select its (Delete) button.
3. Select OK to confirm.
NOTE
The search engine parameter fields vary, depending on the search engine being used, e.g., MaxQuant, Andromeda.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
94

Glossary A
Admin
Role that allows adding plates, creating projects, creating analysis protocols, viewing MS data files, viewing results files, and adding and deleting users.
analysis
A search for identification and annotation of LC-MS data.
Analysis Metrics
Tab that provides the QC metrics for an analysis result.
analysis protocol
The parameters for an MS database search in a .xml or .json file.
Analysis Summary
Tab that provides an overall view of analysis results.
annotation
A highlight or explanatory note added to a chart.
C
CO-RE
Compressed O-ring expansion.
consumables
Reagents and plasticware.
control limits
CV
D
data-dependent acquisition
MS method that derives an MS/MS spectra from selection, isolation, and fragmentation of an individual precursor ion.
data-independent acquisition
MS method that derives an MS/MS spectra from selection, isolation, and fragmentation of all precursor ions in a defined m/z range.
DDA
Data-dependent acquisition.
DIA
Digestion Control
A reference sample added before nanoparticle incubation.
distribution of detected proteins in plasma proteome
The dynamic range of proteins identified in each sample compared to a deep reported human plasma proteome index.
E
equipment
Reusable laboratory equipment.
H
hierarchical clustering
Cluster analysis based on agglomerative nesting, which groups samples in clusters based on similarity.
Parameters that help determine whether results are expected. The mean provides a historical average, and the upper and lower control limits indicate normal variation.
custom file
Optional sample information in a .csv, .tsv, .xls, or .xlsx file.
Coefficient of variation.
Data-independent acquisition.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 95

Glossary
IN
intensities
Protein and peptide intensities and the distribution of protein sequence coverage for each sample, including the coefficient of variation.
NCT
Nested conductive tips. Nanoparticle.
Nested tip rack.
P
PC1
PC2
Principal component 2.
PCA
Principal component analysis.
PCA analysis
Clusters of similar samples based on PC1 and PC scores.
PCC
Pearson correlation coefficient.
Pearson correlation coefficient
A measure of the linear correlation of data.
peptide counts
The number of peptides in each sample.
peptide counts distribution
Peptide counts for the five nanoparticles processed with the samples.
peptide counts of nanoparticles
Sample counts plotted by nanorparticle.
plate
The samples, controls, and nanoparticles from a Proteograph Assay method run.
L
NP NTR
lamppost protein concentration
The intensity of landmark proteins in each sample.
LC
LC-MS
Liquid chromatography-mass spectrometry.
LCL
Lower control limit.
M
Mass Spec Control
Reference peptides added before LC-MS analysis.
materials
Consumables and equipment.
MPE
Monitored multi-flow positive pressure evaporative extraction.
MPE Control
Reference peptides added before desalting cleanup. Mass spectrometry.
MS data file
The results of MS analysis for each sample or control in a plate in a .raw, .wiff, .wiff.scan file.
MS
Liquid chromatography.
Principal component 1.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
96

Glossary
plate map file
The location of each sample in a plate in a .csv file.
plate map grid
Metrics for each sample in the format of 96-well plates.
Plate Samples
Table listing all samples in a plate selected on the Plates page.
Plates
Table on the Plates page listing all plates in PAS.
Process Control
A reference sample added before nanoparticle incubation.
project
All samples in an analysis with sample information.
Projects
Table on the Projects page listing all projects in PAS.
protein group counts
The number of proteins in each sample.
protein group counts distribution
Protein group counts for the five nanoparticles processed with the samples.
protein group overlap sets
Protein group intersections, including intersection size and protein group counts.
Proteograh Analysis Suite
Seer software used to process, analyze, and visualize LC-MS data.
Proteograph
Descriptive product term.
Proteograph Assay Kit
The reagents and labware for preparing samples on the SP100.
Proteograph Instrument Control Software
Software onboard the SP100 used to operate the instrument.
Proteograph Product Suite
The bundle of Seer kit, instrument, and analysis software.
Protocols
Table on the Protocols page listing all analysis protocols in PAS.
Q
QC
QC charts
Charts presenting process control data for an analysis.
QC metrics
Metrics indicating how well an analysis performed.
R
report
A summary of control results in a .pdf file.
results file
The output of an analysis in .txt or .xml file format.
S
sample comparability
The degree of statistical correlation between samples based on the Pearson correlation coefficient or Jaccard index.
sample description file
Metadata for each sample in plate in a .csv file.
Sample list
List of all samples in a project selected on the Projects page.
Seer AutoUploader
An application that runs on the MS computer and automatically transfers new MS data files to PAS.
Quality control.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
97

Glossary
SP100 Automation Instrument
The Seer liquid handling instrument.
summary
A list of each QC metric with the control, well, plate, value, and result in a .csv file.
U
UCL
Upper control limit.
User
Role that allows adding plates, creating projects, creating analysis protocols, viewing MS data files, and viewing results files.
user group
A group of users that can access and view the plates, projects, and analyses of all other users in the group.
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 98

Index
A
accounts 9, 14, 21 add
users 21 adding
custom table columns 12 adjusting tables 77 Analyses page 9, 34, 36, 86 analysis
DDA-based 92 DIA-based 92
metrics 44
performance 44
results 20, 37, 44 settings 20, 54
starting 26, 29, 79-80, 83 status 86
summaries 37 analysis interpretation 60 analysis logs 86-87 analysis metrics 34, 36 Analysis Metrics
Hierarchical Clustering section 52 Intensities section 44-45
Lamppost Proteins’ Concentration sec-
tion 46-47
PCA Analysis section 48
Peptide Counts Distribution section 49 Peptide Counts of Nanoparticles sec-
tion 50-51
Plate Map Grid section 46
Protein Group Counts Distribution sec- tion 49
Protein Group Counts of Nanoparticles section 50-51
Set Plate Map Grid section 45 analysis output files 59
Analysis Output Files tab 59 Analysis Protocol page 9, 92 analysis protocols 15, 19, 37, 79, 92
creating 92-94 defaults 19 parameters 92-94 selecting 31
analysis summary 34, 36 Analysis Summary
Distribution of Detected Proteins in Plasma Proteome section 39-
40
Peptide Counts section 38-39 Protein Group Counts section 37-38 Protein Group Overlap Sets
section 40-41
Sample Comparability section 42 Summary section 37
annotations 9, 39-40, 57 appearance, user interface 19 applications 91
assay controls 9
auto updater 91 B
bar graphs 40 box plots 44, 49 Box Plots 73 browsers 10
C
calculated control limits 55 calculations 39, 45, 55-56, 92 Chart Type preference 45 charts 44, 53
citations 39, 92
Clustered Heatmap 68 clusters 48, 52
coefficient of variation 44, 49 color codes 42, 55-56
Color Labels By preference 43 color scheme
PCA Analysis 48 columns
custom 16
descriptions 76, 83
plate map files 15
required 25, 28, 79 sample description files 16 showing and hiding 46 sorting 77
common proteins 37 comparing samples 52 compatible browsers 10 contact information, Seer 102 control charts 53, 55, 57 control software 15
controls
analyzing 27, 30-31 information 53 metrics 55, 57-58 results 53, 55, 57-58 types 15, 18, 53-54
Coverage 70
creating folders 90
creating projects 26, 29, 79-80, 84 cumulative protein groups 37 custom analysis protocols 92-94 custom columns 16
custom table columns 12 customer support 102
D
dark theme 19 data
distribution 44-45 hiding 55 reanalyzing 24, 31
Data Files page 9, 28, 89 defalt control limits 55 delete
users 22
deleting annotations 57 deleting folders 91 dendrograms 52
density 44
density plots 44-45
detected proteins 37, 39, 47 differences in proteins between
groups 66 digestion controls 18, 54
directories 28, 83, 89
disable authenticator app security 20 Display Percentage preference 41 Display Top N Intersection preference 41 Display Type preference 46
Distribution of Detected Proteins in
Plasma Proteome 39-40 distributions 44-45
E
edit
users 22
emails 19 EncyclopeDIA 92 Enrichment 72
F
field descriptions 76, 83 fields, custom 12
file extensions 15
file formats 15, 17, 87 file management 28, 89 file names 15
Filtered PPI Network 71 filters 53-56, 77, 89 folders 28, 89-91 functions 9, 41
G
Graph Colors preference 40
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
99

Index
Graph Types preference 46 graphs 20
group analysis
differences between groups 66 Group Analysis
Box Plots section 73
Clustered Heatmap section 68 Coverage section 70 Enrichment section 72
Filtered PPI Network section 71 Raw Data section 66
Volcano section 69
Group Analysis tab 60, 66 grouping data 20
H
Heatmap 67
help, technical 102
Hierarchical Clustering 52
historical means 56
Horizontal Bar Position preference 41
I
icons 86
ICS 15
identification rates 45
index 39
injection 54-55
input files 24-25, 28, 79, 82, 93 instruments 15, 45, 54, 57-58, 76 intensities 39, 46
Intensities 44-45
Intensity Plot 67
interpreation of analysis results 60 intersections 40
J
Jaccard index 42-43 L
labels 37-38, 44
Lamppost Proteins’ Concentration 46-47 landmark proteins 46-47
layouts 37, 44
LC-MS 9, 45
LCLs 56
light theme 19
line graphs 37-38, 46
link, PAS 10
links 19
log10 39, 46
logs 86-87
lower control limits 56
M
mass spec controls 18, 54, 56 matrices 40, 42
MaxQuant 92
MaxQuant analysis protocol 92 means 37, 49, 56
method runs 76
metrics, displaying 34, 36, 46 miscleavage rates 45 moving files 89
MPE controls 18, 54
MS computer 91
MS controls 18, 54
MS data files 19, 28, 31, 40
automatic upload 91
file formats 15
file names 15
links 19, 24, 89
number of 86
removing 19, 25, 28, 79, 90
MS database 15, 39, 45, 92
MS file’s name length preference 41
N
n-values 39 nanoparticles 49-50, 76 navigation 9
O
optional inputs 26-27, 29-31, 57 orange, meaning 55
outliers 56
output files 57-59
oxidation ratios 45 P
Page Chart Layout preference 40, 42, 45- 46
pages
Analyses 86
Data Files 89
Plates 76
Projects 83
Protocols 92
Users and Permissions 21
parameter files 93 PAS link 10
PC1 48
PC2 48
PCA Analysis 48
PCC 42
Pearson correlation coefficient 42 Pearson’s correlation coefficient 43 Peptide Collection Plate
contents 76
peptide counts 50
Peptide Counts 38-39
Peptide Counts Distribution 49 Peptide Counts of Nanoparticles 50-51 peptides 37-38, 45
performance 44, 55
permissions 21
Plate Grid section 81
plate map files 15, 79
Plate Map Grid 46
Plate Samples tables 77, 80
plates
contents 15, 76 naming 26, 29, 77 removing 77
Plates page 9, 25, 76 Plates table 76
PPI Network 71 preferences
Distribution of Detected Proteins in Plasma Proteome 40
Intensities 45
Lamppost Proteins’ Concentration 47 PCA Analysis 48
Peptide Counts 39
Plate Map Grid 46
Protein Group and Peptide Counts of
Nanoparticles 51 Protein Group Counts 38
Protein Group Overlap Sets 41
Sample Comparability 42 principal component analysis 48 process controls 18, 53-54 projects
creating 26, 29, 79-80, 83-84 removing 84
revising 85
Projects page 9, 31, 83
Projects table 83
Protein Group Counts 37-38
Protein Group Counts Distribution 49 Protein Group Counts of
Nanoparticles 50-51 Protein Group Overlap Sets 40-41 protein groups 37, 40, 45
comparisons 42
intersections 40 protein names 40, 46-47 proteins 39
adding 47 annotated 40 counts 49 distribution 39-40 highlighting 40
Proteins preference 40 Proteograph Assay method
output 76
Proteograph Instrument Control
Software 15 proteome 40
Protocols page 92
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5)
100

Index
Q
QC charts 34, 54-55
quality control 44, 47, 54, 66 quantification 45
quantiles 40, 44
Quantiles preference 40 quartiles 44-45
R
ranges, control limits 56 Raw Data 66
reanalyzing data 24, 31
red proteins 39
reference peptides 18 reference samples 18 references 39, 92
renaming files 89
renaming folders 90 required columns 25, 28, 79 results 9, 34, 36
results files 17, 34
results, interpretation of 60 runs 37, 76
S
Sample Comparability 42
sample description files 15, 79-80 sample information 16, 26, 29, 37, 82-83 samples
adding 83
analyzing 27, 30-31 correlations 42
grid view 81
nanoparticles 50
number of peptides 38, 45 number of proteins 39, 45 reviewing 77, 81-82 similar 48, 52
table view 81
92 sidebar 9
Similarity Score preference 43 similarity scores 42
sorting 20, 77
starting analysis 26, 29, 79-80, 83 starting points 25, 28, 31
status, analysis 86 summaries 53, 57-58 Summary 37
T
table view 82 tables
adjusting 77
teal, meaning 55-56
technical assistance 102
templates 25, 28, 79
tenants 14
theme 19
trends 37
turn two-factor authentication on or off 20 two-factor authentication 19
U
UCLs 56
upper control limits 56 user groups 55
user interface 9
customizing 19-20, 37, 41, 44 light or dark 19
navigation 9
scaling 46
User menu 21
user roles 21
Users and Permissions page 21 Users menu 21
Users page 9, 21
V
Vertical Height Rate preference 41 violin plots 44-45
Volcano 69
X
X Tick Label preference 38-39 Y
Y Axis Range preference 38-39, 51, 55 yellow wells 77, 81
scaling 46
Scaling preference 46
scatter plots 48, 50
search engine 9, 92
search engine parameters 93
Search field 21, 66, 76, 78, 83-84, 86, 92 search parameters 15, 92
search results 17
Seer contact information 102
Set Plate Map Grid 45
settings 37, 40, 44, 54-55
Default Analysis Protocol 19
Receive email notifications on analysis
updates 19
Remove MS data files when plate is
deleted 19
Use Authenticator App 19
Show/Hide dropdown 76, 78, 83-84, 86,
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 101

Technical Support
For technical assistance, contact Seer.
Contact Information
Seer, Inc.
3800 Bridge Pkwy #102 Redwood City, CA 94065 Email: support@seer.bio
seer.bio
Telephone
650.453.0000
Proteograph Analysis Suite User Guide (CF-1003 B , PAS version 1.5) 102

Seer, Inc.
3800 Bridge Pkwy #102 Redwood City, CA 94065 650.453.0000 info@seer.bio
seer.bio