Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics

Historically, proteomes, the intricate landscape of proteins within our bodies, have been challenging to explore and interpret. With their complex biochemistry governed by a kaleidoscope of post-translational modifications, multiple protein-protein interactions, and wide dynamic range, unlocking their important biological ‘secrets’ has been no small feat. Yet, it is precisely within these proteomes that we can uncover critical information about health, disease, and other biological states.

Seer’s Proteograph™ XT Assay nanoparticle-based workflow is purpose-built to conquer the inherent challenges of proteomics research. Its cutting-edge technology defies the odds by compressing the dynamic range at the nano-bio interface, allowing unprecedented deep access to the plasma protein on a scale like never before.

Here, in this pilot study assessing the Proteograph XT workflow’s quantification performance, we showcase the true extent of its capabilities, particularly when combined with Thermo Fisher’s high-speed and highly-sensitive Orbitrap Astral™ Mass Spectrometer, to facilitate high-throughput, deep, and quantitative plasma proteomics and uncover biomarker signatures at peptide level resolution.

While 22 proteins monopolize 99% of the protein mass in plasma, the remaining 1% harbors thousands of lesser-known players – low-abundance proteins and their variants – making it challenging for researchers seeking to dive deep into the plasma proteome, without sacrificing coverage and throughput.

The Study Design

We evaluated the Proteograph XT workflow’s quantification performance by assessing relative fold-change accuracy and linearity using the gold-standard – multi-species proteome spike-in experiment. To evaluate both large and small fold-changes, bovine plasma samples were mixed with two pooled human plasma samples at seven different Bovine:Human ratios. The Bovine:Human mixed sample data was assessed using the Proteograph XT workflow and the reference neat plasma digestion workflows. This data was also used to examine the quantification precision across the 200-subject AD cohort (> 1000 MS injections) to determine the statistical power of the Proteograph XT workflow in an example plasma cohort study.

Spike-in experiment

Bovine plasma samples were spiked into two different pooled human plasma samples at seven different Bovine:Human ratios. (1:0, 1:1, 1:1.5, 1:2, 1:9, 1:99, 0:1.)

Reproducibility study

A control pooled human plasma sample was processed with two different Proteograph SP100 Automation Instruments on two separate days each, resulting in a total of 4 batches (plates).

Cohort study

A pooled human plasma sample was processed as a process control on each Proteograph XT Assay plate and then analyzed across the 200 plasma cohort samples, resulting in 117 quality control measurements.

The Results

The study demonstrated the ability of the Proteograph XT workflow to quantify peptides and proteins with unprecedented scale and precision, independent of the chosen linearity, fold change accuracy, or precision level.  Offering a new gateway to the proteome, Seer’s nanoparticle-based approach provided enhanced visibility via downstream MS detectors, capturing quantitative differences across samples with greater numbers of proteins and peptides. Over 6,000 protein groups from protein plasma were identified, revealing the true power of this workflow and offering researchers the exciting opportunity to dive deeper into the plasma proteome and usher in a new era of biological insights.