Last Updated
11 March 2021

AACR: Identification of Differential Plasma Protein Isoforms in Early Lung Cancer

Proteograph Platform Delivers Unbiased, Deep and Rapid Proteomics at Scale
The ~20,000 genes in the human genome encode over one million protein variants, because of alternative splice forms, allelic variation and protein modifications. Though large-scale genomics studies have expanded our understanding of cancer biology through analysis of both tissue and biofluids, similarly-scaled unbiased, deep proteomics studies of biofluids have remained impractical due to complexity of workflows. W e have previously described the Proteograph Product Suite, a novel platform that leverages the nano-bio interactions of nanoparticles for deep and unbiased proteomic sampling at scale. We have shown the utility of the Proteograph solution for unbiased and deep interrogation of plasma from 141 subjects: 80 pre-classified healthy controls and 61 samples from early-stage NSCLC patients to create a plasma biomarker classifier for the detection of NSCLC versus healthy
controls with AUC of 0.91 . Here we present a further analysis of this
data to dissect differences between patients and controls in plasma abundance of protein isoforms arising from alternative gene splicing.
Utilizing Proteograph Platform to Interrogate Protein Isoforms in a Non-small Cell Lung Cancer (NSCLC) Plasma Proteome Study
Proteograph Platform Enables Deep and Unbiased Plasma Proteomics
Proteograph Data Sheds Light on Biological Consequences of Protein Isoforms
BMP1 Shows Differential Isoform Abundance Pattern
The lowest abundant putative protein isoform, BMP1 from the list of candidate proteins identified in this study, comprises four protein coding isoforms. Two of these isoforms are substantially longer (~400-800 residues) than the other two isoforms covering additional exons. Peptides mapping to exons that cover all four protein isoforms (5A) have higher abundance in cancer relative to controls, whereas peptides mapping to exons that cover only the two longer isoforms have higher abundance in healthy controls. BMP1 is known to play a dual role in cancer, acting as both suppressor and activator3 and this differential pattern of isoform abundance may shed further light into BMP1’s role in cancer.
1,000 800 600 400 200 0
Static indicator of risk
High accessibility of content but low utility
Utility of Information 10M+ human exomes
1M+ genomes
Dynamic indicator of status
High utility but low accessibility of content
~695M genetic variants catalogued
13467 25
13467 25
Figure 2. Overview of the putative protein isoform identification strategy. From 1,992 proteins we filtered to proteins present in at least 50% of subjects from either heathy or early cases and searched for peptides that had differential abundance between controls and cancer (p < 0.05; Benjamini- Hochberg corrected). Next, we filtered for proteins comprising sets of peptides where at least one peptide had significantly higher and another significantly lower plasma abundance in healthy controls vs. early NSCLC. Figure 5. BMP1 exon map and peptide intensity. BMP1 peptides from NSCLC early and control subjects mapped to four BMP protein- coding transcripts (5A) and corresponding peptide intensities (5B) shows; two of the peptides (purple shading) are more abundant in NSCLC and five of the peptides (teal shading) are more abundant in healthy controls. We demonstrated that measurements at the peptide level for the plasma proteome enable quantification of differential isoform abundance patterns, which were inaccessible to prior methods of lesser scale, depth, or coverage compared to the Proteograph platform. By extending our approach to include additional features such as protein amino acid variants and PTMs, we anticipate extending our knowledge to enable proteogenomics. The Proteograph Product Suite has the throughput required for the identification of protein isoforms across the proteome in an unbiased manner, enabling cancer biology research and biomarker discoveries at scale. References: 1. Blume et al. Nat. Comm. (2020) 2. Deutsch et al. J. Proteome Res.(2018) 3. Bach et al. Mol. Ther. Oncolytics (2018 Plasma Data analysis Proteins MaxQuant Nanoparticles Protein coronas Digestion 5ug < 0.2% of genetic variants fully characterized 1.0 0.5 0.0 Detected Peptides Protein X *** Peptide *** Peptide *** Peptide Core Technology Proprietary Engineered Nanoparticles Peptide Peptide Peptide Peptide 6 5 4 3 2 Microflow SWATH LC/MS analysis Tryptic peptides APOB RAP1B VCL TLN1 FLNA BMP1 COL6A3 PRG4 LDHB RTN4 FERMT3 HADHA THBS3 ITIH1 C4A C1R 16 candidate proteins 0.4 0.6 0.8 Proteograph Product Suite Sample is ready to be analyzed on most LC/MS instruments Figure 3. Identification of 16 putative protein isoforms and corresponding open targets score. Associated open targets score for lung carcinoma targets with putative protein isoforms. Nine novel lung carcinoma targets with little or non-existing information are highlighted (teal bracket). Seer, Inc., Redwood City, CA - * © Seer 2021, Proteograph and Seer are trademarks of Seer Inc, all other trademarks are property of their respective owners Application of the ProteographTM Product Suite to the Identification of Differential Protein Isoform Plasma Abundance in Early Lung Cancer vs. Healthy Controls Asim Siddiqui*, John E. Blume, Margaret K. R. Donovan, Marwin Ko, Ryan W. Benz, Theodore L. Platt, Juan C. Cuevas, Serafim Batzoglou and Omid C. Farokhzad Percentage of Samples in Which Protein Group Detected 2499 1992 Figure 1. NSCLC study using the Proteograph Product Suite. Number of subjects in NSCLC study including healthy controls and early NSCLC samples (1A). Protein groups detected across 141 subjects in control vs. early NSCLC plasma samples (1B), across a percentage of the subjects in the NSCLC study. 2499 protein groups are found across all subjects and 1,992 in 25% of the subjects. Number of peptides for each protein group in the NSCLC study (1C) shows, 21,959 peptides are detected in total with a median 8 peptides per protein across the NSCLC plasma study using the Proteograph Product Suite. 1A 1B n=80 n=61 Sample Type 1C Identification of Putative Protein Isoforms Using Peptide Abundance 2 2 2 2 p = 6.6e−03 0.0 0.2 16 Candidate Proteins 16 Candidate Proteins Ranked by the HPPP 15/3486 Matched Intensity Rank Figure 4. Putative protein isoforms matched to the Human Plasma Proteome Project (HPPP)2. 15 out 16 proteins were found in the HPPP. These were ranked by estimated concentration in plasma. P13497 (BMP1) is the least abundant. P61224 (RAP1B) is not present in the HPPP. Associated Open Targets Score Associated Open Target Score Number of Peptides Detected per Protein Median 8 5A BMP-202 ENST00000306385 Peptide 7 Peptide 6 Peptide 4/5 Peptide 3 Peptide 2 Peptide 1 BMP-201 ENST00000306349 BMP-204 BMP-203 ENST00000354870 5B ENST00000397814 Peptide 1 p = 2.1e−04 Peptide 3 Peptide 4 p = 4.8e−02 Peptide 5 p = 1.1e−01 Peptide 6 p = 5.8e−02 Peptide 7 p = 3.5e−02 1 1 1 1 1 Peptide 2 p = 2.7e−02 346 25 BMP1 BMP1 Early NSCLC Healthy Control 1346 25 Estimated ng/mL NSCLC Control Cataloged variants (mm) Early NSCLC Healthy Early NSCLC Healthy Early NSCLC Healthy Early NSCLC Healthy Early NSCLC Healthy Early NSCLC Healthy Early NSCLC Healthy Peptides Intensity Relative Abundance Early NSCLC Control (Healthy) Early Healthy Early Healthy Early Healthy Early Healthy Number of Subjects Protein Group Counts Number of Proteins