Accelerating Throughput with the Proteograph? XT Assay

Plasma proteomics holds extraordinary potential for helping evaluate human health and disease, including the early detection of life-threatening conditions such as cancer.

Yet the extensive dynamic range and diversity of protein variants in plasma presents unique challenges when it comes to achieving the simultaneous depth of coverage and throughput needed for large-scale plasma proteome studies. With traditional methods, researchers have to make a trade-off, which limits their ability to perform in-depth proteomic studies at scale.??

As the world continues to witness a paradigm shift in proteomics research, our Proteograph? XT Assay is helping transform it by facilitating deep and unbiased proteomics at enhanced throughput. Enabling the reproducible quantification of thousands of proteins in large cohort plasma studies, without compromising depth and while enhancing the throughput of proteomics analysis,?the Proteograph XT workflow is creating a novel opportunity to detect protein biomarkers in ways like never before.??

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KEY INSIGHTS

• The Proteograph XT Assay empowers large-scale cohort studies while preserving optimal coverage, depth, and reproducibility.??
• In a study of ~1,800 samples, over 5,000 plasma proteins were identified with the library-free search across samples - with a throughput of one hour per sample, LC-MS analysis time.?
• The end-to-end workflow, encompassing sample processing and LC-MS analysis, was successfully executed within a span of around 11 weeks.
• The Proteograph XT Assay's coverage of the Human Plasma Proteome Project (HPPP) database highlights its unmatched potential to decipher depth in plasma proteome large-scale plasma studies.??

THE STUDY DESIGN

In this study, our objective was to evaluate the performance of the Proteograph XT Assay workflow - followed by a 1-hour LC-MS sample analysis throughput with the data independent acquisition (DIA) liquid chromatography mass spectrometry (LC-MS) method - across a cohort of ~1,800 human plasma samples.??

We assessed three library search methodologies and evaluated peptide and protein identification rates, data missingness, depth of coverage, reproducibility, and statistical power analysis.??

Samples were processed by running 47 Proteograph XT Assay plates across three SP100 Automation Instruments, followed by DIA LC-MS analysis of tryptic peptides on one Orbitrap Exploris 480 MS. The data was then analyzed using the Proteograph Analysis Suite (PAS).?

THE RESULTS

The Proteograph XT Assay workflow with the Orbitrap Exploris 480 MS by Thermo Fisher Scientific, demonstrated unprecedented depth, reproducibility, and throughput - providing researchers with the ability to amplify their proteomics research discovery power.?

When comparing the DIA LC-MS results from library-free, experiment-specific gas-phase fractionation (GPF) and our PAS default library search, we were able to demonstrate that while library-free searches identify more protein groups, an experiment-specific or the PAS default library results in improved data completeness – providing?researchers with the ability?to conduct higher powered studies.?

1. Protein Group and Peptide Identification?- We identified 5,243 protein groups and 53,640 peptides across all samples using the library-free search, 2,892 protein groups and 25,445 peptides using the PAS default library, and 4,007 protein groups and 36,259 peptides using the GPF library.?
2. Reproducibility?- In the realm of discovery proteomics, assay reproducibility and accuracy go hand in hand. The results illustrated that biological variation surpassed technical variation, even when involving subtle protein changes in blood plasma, indicating the potential to detect discernible biological differences among samples. ? ?
3. Power Analysis?- The technical reproducibility of the workflow, especially when surpassing technical variation, provides a new technology for researchers in the field of protein biomarker discovery.???
4. Depth?- One of the greatest challenges in proteomics is addressing the dynamic range of the plasma proteome. The plasma proteome spans over 12 orders of magnitude, which is beyond the range detectable by modern LC-MS detection limits. To assess the extent to which the Proteograph XT Assay enables the assessment of proteins across the wide dynamic range of the plasma proteome, we mapped the identified proteins to the Human Plasma Proteome Project (HPPP) database. Our observation was these proteins span nine orders of magnitude and the entire reported abundance range of HPPP, from highly abundant P02768 (7 x 105 ng/mL, ALB) to lowly abundant Q63HN8 and P46939 (0.004 ng/mL; RNF213 and UTRN).??

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