Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

American Chemical Society full text link American Chemical Society Free PMC article
Full text links

Actions

Review
.2023 Mar 3;22(3):697-705.
doi: 10.1021/acs.jproteome.2c00721. Epub 2023 Feb 3.

Strategies for Increasing the Depth and Throughput of Protein Analysis by plexDIA

Affiliations
Review

Strategies for Increasing the Depth and Throughput of Protein Analysis by plexDIA

Jason Derks et al. J Proteome Res..

Abstract

Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this potential requires sensitive and deep protein analysis of a large number of samples. Toward this goal, proteomics throughput can be increased by parallelizing the analysis of both precursors and samples using multiplexed data independent acquisition (DIA) implemented by the plexDIA framework: https://plexDIA.slavovlab.net. Here we demonstrate the improved precisions of retention time estimates within plexDIA and how this enables more accurate protein quantification. plexDIA has demonstrated multiplicative gains in throughput, and these gains may be substantially amplified by improving the multiplexing reagents, data acquisition, and interpretation. We discuss future directions for advancing plexDIA, which include engineering optimized mass-tags for high-plexDIA, introducing isotopologous carriers, and developing algorithms that utilize the regular structures of plexDIA data to improve sensitivity, proteome coverage, and quantitative accuracy. These advances in plexDIA will increase the throughput of functional proteomic assays, including quantifying protein conformations, turnover dynamics, modifications states and activities. The sensitivity of these assays will extend to single-cell analysis, thus enabling functional single-cell protein analysis.

Keywords: high-throughput; isotopologous carriers; mass tags; multiplexed data independent acquisition; multiplexed proteomics; plexDIA; sensitive proteomics; single-cell proteomics.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Parallel precursor isolation and fragmentation enableanalyzingall detectable precursors even when using long ion accumulation timesfor MS2 scans.a, As ion accumulation times increase,the number of precursors that can be fragmented and analyzed at MS2level decreases for data dependent acquisition (DDA) analysis. TheDDA graphs show a theoretical estimate for the maximum number of precursorsthat can be analyzed as a function of ion accumulation times for MS2scans while using a 60 min active gradient and assuming full dutycycles. In contrast, parallel isolationand fragmentation of precursors by DIA allows for analyzing all detectableprecursors even when using long ion accumulation times for MS2 scans.b, As accumulation times increase, the points per elutionpeak decrease as illustrated for duty cycles having 10 MS2 scans percycle and either 1 or 3 MS2 scans per duty cycle. Elution peaks weremodeled as 20 s at base for 60 min active gradient, and narrower peakswill have fewer sampling points.
Figure 2
Figure 2
The accuracy of proteinquantification at MS2-level increases withthe translation algorithm.a, plexDIA uses a translationalgorithm to reduce the impact of interferences by scaling the apexesof fragments from propagated sequences to the most confident sequence. The algorithm uses the average fragment ratioto scale the quantity of the best quantified precursors to other precursorswith the same sequence. This panel was adapted from Derks et al.b, Mixed species samples used tobenchmark plexDIA performance were usedto assess quantitative accuracy with and without translation. Boxplotdistributions of MS1 and MS2-level deviations from the expected ratiosare plotted for the precursor ratios (n = 37,907)that were quantified in common across all samples. MS2-level quantitativeaccuracy improves in the “MS2 translated” condition(orange) relative to nontranslated quantities (yellow).
Figure 3
Figure 3
Precision of retentiontime estimates within and between plexDIAruns.a, Extracted Ion chromatogram (XIC) for the precursorsof two peptides quantified in U937 and PDAC cells. Circles correspondto measured intensities and the curves are colored based on the intensity.b, The retention time (RT) deviations are estimated from triplicateinjections of plexDIA samples composed of 100 cells of Melanoma, PDAC,and U-937 cells and analyzed with high frequency survey scans. ApexRTs within a run between channels comparing PDAC and U937 cells (“Withinrun”) are more similar than the aligned RTs (“Betweenruns”). The median absolute RT deviations (|ΔRT|) areindicated on top of each distribution in milliseconds (ms).
See this image and copyright information in PMC

References

    1. Cravatt B. F.; Simon G. M.; Yates J. R. 3rd The Biological Impact of Mass-Spectrometry-Based Proteomics. Nature 2007, 450 (7172), 991–1000. 10.1038/nature06525. - DOI - PubMed
    1. Heck A. J. R.; Krijgsveld J. Mass Spectrometry-Based Quantitative Proteomics. Expert Rev. Proteomics 2004, 1 (3), 317–326. 10.1586/14789450.1.3.317. - DOI - PubMed
    1. Zhang Y.; Fonslow B. R.; Shan B.; Baek M.-C.; Yates J. R. 3rd Protein Analysis by Shotgun/bottom-up Proteomics. Chem. Rev. 2013, 113 (4), 2343–2394. 10.1021/cr3003533. - DOI - PMC - PubMed
    1. Gatto L.; Aebersold R.; Cox J.; Demichev V.; Derks J.; Emmott E.; Franks A. M.; Ivanov A. R.; Kelly R. T.; Khoury L.; Leduc A.; MacCoss M. J.; Nemes P.; Perlman D. H.; Petelski A. A.; Rose C. M.; Schoof E. M.; Van Eyk J.; Vanderaa C.; Yates J. R. III; Slavov N. Initial Recommendations for Performing, Benchmarking, and Reporting Single-Cell Proteomics Experiments. Nature methods 2023, in press10.1038/s41592-023-01785-3. - DOI - PMC - PubMed
    1. Virant-Klun I.; Leicht S.; Hughes C.; Krijgsveld J. Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes. Mol. Cell. Proteomics 2016, 15 (8), 2616–2627. 10.1074/mcp.M115.056887. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full text links
American Chemical Society full text link American Chemical Society Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp