MSACL 2019 EU Abstract

DirectMS1: A High-Throughput Analysis for Identification and Quantification of 1000 Proteins in 5 Minutes

Mark V. Ivanov (1), Julia A. Bubis (1), Vladimir A. Gorshkov (2), Irina A Tarasova (1), Elizaveta M Solovyeva (1), Lev I. Levitsky (1), Anna A Lobas (1), Marina L Pridatchenko (1), Frank Kjeldsen (2), Mikhail V. Gorshkov (1)
(1) V.L. Talrose Institute for Energy Problems of Chemical Physics at N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences , Moscow, Russia. (2) Department of Biochemistry and Molecular Biology, University of Southern Denmark Odense M, Odense, Denmark

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Mark Ivanov (Presenter) INEPCP FRCCP RAS

Presenter Bio: My primary background is applied mathematics and physics which was received in Moscow Institute of Physics and Technology (bachelor, master and PhD degrees). I am a proteomics bioinformatician in Mike Gorshkov’s laboratory since 2011. I had a 3-months internship in Renato Zenobi’s lab (ETH Zurich) in 2012. Just finished PhD in the end of 2018. I’m a coauthor of 24 publications in high impact journals. I have an experience as participant of multiple Russian (RFBR, RSCF) and European (FP7) grants.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction:
Bottom-up proteome-wide analysis is based on the separation of proteolytic peptides followed by tandem mass-spectrometry (MS/MS). The latter step includes sequential isolation of the eluting peptides followed by their fragmentation. The needed fragmentation step is the bottleneck of the bottom-up proteomics limiting its use in biomedical applications. The MS/MS-based proteome analysis is slow and require long LC gradients for successful identification of low abundant proteins and providing high sequence coverage for quantification.

Objectives:
The primary objective of this study was to show limits and capabilities of mass-spectrometry based proteomics without using tandem mass spectra.

Methods:
Experiments were performed using high resolution mass-spectrometry Orbitrap (QExactive HF) coupled with Dionex nano-flow HPLC system. 5 minute LC gradients were used for this study. Different amounts of standard HeLa digest starting from as low as 1 ng were loaded for the shotgun analyses and efficiency comparison of proposed MS1-only and MS/MS-based methods. Quantification efficiency was tested on samples prepared in iPRG2015-like manner, where 6 proteins were spiked in yeast background in different concentrations. Data processing for protein identification and quantification which implements proposed MS1-only approach was performed using in-house developed software ms1searchpy and Diffacto software for quantification.

Results:
We report on LC-MS1 based approach outperforming regular data-dependent acquisition in terms of number of identified proteins, limit of detection and quantification accuracy when short LC gradients are used. In this study, up to 1000 protein groups were successfully identified in MS1-only mode for a standard HeLa cell line digest using a 5 minute LC gradient. The amount of loaded sample was varied in a range from 1 ng to 200 ng, and the method demonstrated 10-fold better sensitivity compared with the standard MS/MS-based approach. Comparison of the quantitation accuracy of the methods demonstrated significant outperformance of MS1-only approach over most common MS/MS LFQ methods (NSAF, MaxLFQ, Diffacto).

Conclusion:
Rapid MS1-only proteome characterization can be an alternative to the MS/MS-based “shotgun” analyses in the studies, in which the experimental time is more important than the depth of the proteome coverage.