Accelerated Proteomic Sample Preparation for Accurate Ultrafast Mass Spectrometry-Based Quantitative Analysis of Cell and Tissue Proteomes

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Daria D. Emekeeva^1#, Tomiris Kusainova^1#, Leyla A. Garibova^1#, Andrey A. Shelepchikov², Alexey S. Kononikhin¹, Alexey V. Tretyakov², Olga I. Lavrukhina², Evgeny N. Nikolaev¹, Mikhail V. Gorshkov¹, Irina A. Tarasova^1,a*

¹V. L. Talrose Institute for Energy Problems of Chemical Physics, N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia

²The Russian State Center for Animal Feed and Drug Standardization and Quality, 123022 Moscow, Russia

^* To whom correspondence should be addressed.

^# These authors contributed equally to the work.

Received: April 3, 2025; Revised: May 16, 2025; Accepted: May 17, 2025

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Advances in liquid chromatography/mass spectrometry (LC-MS) have enabled proteome-wide quantitation in minutes, achieving rate of 1000 analyses per day. This necessitates revisiting the rapid sample preparation approaches to match this data acquisition speed. Despite the fact that these approaches have been developed decades ago, their application in quantitative ultrafast proteomics and comprehensive comparison of their performance under different conditions have not been explored. In this study, the ultrasound, microwave irradiation, and elevated temperature-assisted approaches for accelerated protein reduction, alkylation, and trypsin digestion were compared. Validation was carried out with label-free quantitative LC-MS/MS and fragmentation-free DirectMS1 methods of shotgun proteome analyses of Saccharomyces cerevisiae, human cell lines, and winter wheat shoots. These data acquisition methods were applied in ultrafast implementations employing 5 to 16 min LC gradients. Human–yeast proteome mixtures were used as standards to evaluate quantitation accuracy of the sample preparation workflows. Our findings indicate that the reduced time of sample preparation insignificantly decreased efficiency of reduction, alkylation, and digestion, yet, preserved reproducible peptide and protein identification. We also found that the 30-min microwave-assisted and overnight trypsin digestion yielded comparable quantitation accuracy in ultrafast analyses using DirectMS1 method.

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KEY WORDS: ultrafast proteomics, sample preparation, fragmentation-free mass spectrometry, accelerated protein digestion, microwave digestion

DOI: 10.1134/S0006297925600930

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