Accelerated proteomic sample preparation for accurate ultrafast mass spectrometry-based quantitative analysis of cell and tissue proteomes

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Abstract

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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About the authors

D. D. Emekeeva

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

Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

T. T. Kusainova

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

Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

L. A. Garibova

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

Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

A. A. Shelepchikov

The Russian State Center for Animal Feed and Drug Standardization and Quality

Email: iatarasova@yandex.ru
Russian Federation, 123022 Moscow

A. S. Kononikhin

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

Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

V. V. Tretyakov

The Russian State Center for Animal Feed and Drug Standardization and Quality

Email: iatarasova@yandex.ru
Russian Federation, 123022 Moscow

O. I. Lavrukhina

The Russian State Center for Animal Feed and Drug Standardization and Quality

Email: iatarasova@yandex.ru
Russian Federation, 123022 Moscow

E. N. Nikolaev

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

Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

M. V. Gorshkov

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

Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

I. A. Tarasova

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

Author for correspondence.
Email: iatarasova@yandex.ru
Russian Federation, 119334 Moscow

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2. Fig. 1. SDS-PAGE for verification of accelerated hydrolysis protocols. a – Human cell line A549; b – shoots of 7-day-old wheat T. aestivum seedlings; c – baker's yeast S. cerevisiae. Designations: M – molecular weight markers; B – control protein lysates of each organism; 1 (control), 2, 3, 4_37, 4_50 and 5 – peptide probes obtained using hydrolysis protocols 1–5 (Table 1), respectively.

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3. Fig. 2. Number of protein groups identified by HPLC-MS1 (a) and HPLC-MS/MS (b) for A549 cells (H. sapiens); wheat shoots (T. aestivum) and yeast (S. cerevisiae). Each column represents the mean of five independent technical replicates at the sample preparation level. Columns with whiskers represent the mean ± SD in the 95% confidence interval. Asterisks (*) indicate statistically significant changes (Student's t-test; p-value < 0.05) compared to classical hydrolysis (red shaded columns). Abscissa axis legend: 1 – classical 18-hour hydrolysis; 2 – ultrasound exposure at elevated temperature and reduced time of reduction, alkylation and hydrolysis; 3 – hydrolysis at elevated temperature and reduced time of reduction and alkylation reactions; 4_37 – reduced classical hydrolysis; 4_50 – hydrolysis at elevated temperature; 5 – microwave exposure with reduced time of reduction, alkylation and hydrolysis

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4. Fig. 3. Efficiency of enzymatic hydrolysis, reduction and alkylation of cysteine ​​residues for different sample preparation methods based on MS/MS data. a – Number of cysteine-containing peptides relative to the total number of identified peptides for the A549 cell line (Hsapiens), wheat shoots (Taestivum) and yeast (Scerevisiae). b – Relative abundance of peptides with missed hydrolysis sites (0, 1, 2, and 3) calculated from peptide identification in MS/MS data for the A549 cell line (Hsapiens), wheat sprouts (Taestivum), and yeast (Scerevisiae). Data are averaged over five technical replicates for each protocol. Error bars represent the mean ±standard deviation in the 95% confidence interval. Asterisks (*) indicate statistically significant differences (Student's t-test; p-value < 0.05) when compared with the classical protocol (marked with the symbol "0"). Designations on the abscissa axis: 1 - classical 18-hour hydrolysis; 2 - ultrasound exposure at elevated temperature and reduced time of reduction, alkylation and hydrolysis; 3 - hydrolysis at elevated temperature and reduced time of reduction and alkylation reactions; 4_37 - reduced classical hydrolysis; 4_50 - hydrolysis at elevated temperature; 5 - microwave exposure with reduced time of reduction, alkylation and hydrolysis

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5. Fig. 4. Overlapping peptide (a) and protein (b) identifications in replicates at the sample preparation level: MS/MS data analysis. Each protocol was applied to prepare samples from the A549 cell line (H. sapiens), wheat sprouts (T. aestivum), and yeast (S. cerevisiae). Number of replicates: 4 (human) or 5 (wheat and yeast). Key: 1 – classical 18-h hydrolysis; 2 – ultrasound exposure at elevated temperature and reduced time of reduction, alkylation, and hydrolysis; 3 – hydrolysis at elevated temperature and reduced time of reduction and alkylation reactions; 4_37 – reduced classical hydrolysis; 4_50 – hydrolysis at elevated temperature; 5 – microwave exposure with reduced recovery, alkylation and hydrolysis time

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6. Fig. 5. Evaluation of the effect of microwave-assisted sample preparation protocol on the results of label-free analysis of quantitative yeast–human protein mixture standards: HPLC-MS1 analysis data. a – Number of identified proteins in human–yeast standards subjected to enzymatic hydrolysis according to the classical (18-hour) protocol 1 (red columns) and the microwave-assisted protocol (purple columns). Designations on the abscissa axis correspond to the microwave-assisted protocol (MW_) or the classical protocol (CL_), as well as to the names of samples with the component ratio (N, N = 1–4) from Table 2. b – Percentage of identified yeast protein groups in human–yeast mixtures compared to the percentage of yeast mass in quantitative standards (shown by dotted lines). Whiskers represent mean ± standard deviation at 95% confidence interval; data are averaged over four replicates (c–h). Scatterplots for pairwise comparison of human–yeast quantitative standards (N2/N1, N3/N1, N4/N1; Table 2) prepared using the classical protocol (c–d) and the microwave-treated protocol (e–h). Dashed brown lines indicate the threshold for selection of differentially regulated proteins. Solid green lines show the actual fold change in protein concentration (FC; log2 scale) calculated using the yeast masses from Table 2. Human and yeast proteins are shown as red and purple markers, respectively

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