The Impact of Different Alkylation Quenching Methods on Tryptic Activity and Protein Identification in Proteomics Sample Preparation
Yuan Gao
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorMin Wang
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorLulu Wang
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorXinglong Jia
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorChunqiu Hu
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
College of Pharmacy, Jiangsu Ocean University, Lianyungang, Jiangsu, China
Search for more papers by this authorPing Liu
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorBin Liu
College of Pharmacy, Jiangsu Ocean University, Lianyungang, Jiangsu, China
Search for more papers by this authorMinjia Tan
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
College of Pharmacy, Jiangsu Ocean University, Lianyungang, Jiangsu, China
Search for more papers by this authorCorresponding Author
Linhui Zhai
Translational Research Institute of Brain and Brain-Like Intelligence, Shanghai Fourth People's Hospital, School of Medicine, Tongji University, Shanghai, China
Correspondence:
Linhui Zhai ([email protected])
Search for more papers by this authorYuan Gao
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorMin Wang
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorLulu Wang
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorXinglong Jia
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorChunqiu Hu
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
College of Pharmacy, Jiangsu Ocean University, Lianyungang, Jiangsu, China
Search for more papers by this authorPing Liu
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Search for more papers by this authorBin Liu
College of Pharmacy, Jiangsu Ocean University, Lianyungang, Jiangsu, China
Search for more papers by this authorMinjia Tan
School of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
College of Pharmacy, Jiangsu Ocean University, Lianyungang, Jiangsu, China
Search for more papers by this authorCorresponding Author
Linhui Zhai
Translational Research Institute of Brain and Brain-Like Intelligence, Shanghai Fourth People's Hospital, School of Medicine, Tongji University, Shanghai, China
Correspondence:
Linhui Zhai ([email protected])
Search for more papers by this authorFunding: This work was supported by grants from the National Natural Science Foundation of China (32171434 and 22225702), the Program of Shanghai Academic Research Leader (No. 22XD1420900), and the State Key Laboratory of Drug Research (SIMM2105KF-13).
Yuan Gao, Min Wang, and Lulu Wang contributed equally.
ABSTRACT
The reduction and alkylation steps are crucial in shotgun proteomics sample preparation to ensure efficient protein digestion and prevent the reformation of artefactual disulfide bonds following proteolysis. Excessive alkylation reagents can lead to overalkylation side reactions, compromising the quality of proteomics sample detection. Previous research has predominantly focused on comparing the effects of various types or concentrations of reducing agents or alkylating reagents for proteomic sample preparation. However, there is a lack of studies systematically comparing the utilization of quenching agents for alkylation reactions and investigating their specific impact on tryptic digestion activity in proteomics sample preparation under conditions of excessive alkylation reagents. In this study, we comprehensively compared the impacts of three different alkylation quenching methods (including cysteine quenching, dithiothreitol [DTT] quenching, and no quenching) on proteomic sample preparation. The upstream sample processing included reduction with DTT or tris(2-carboxyethyl)phosphine (TCEP), followed by alkylation with iodoacetamide (IAA) or chloroacetamide (CAA). Our study demonstrates that the choice of quenching method significantly affects the number of identified proteins and peptides, missed cleavage rates at lysine or arginine residues during trypsin digestion, and the occurrence of overalkylation side reactions. Importantly, our findings indicate that cysteine quenching effectively preserves trypsin activity, ensuring high-quality protein sample preparation. This study provides a systematic analysis of various alkylation quenching methods in proteomic sample preparation and offers optimized experimental protocols and valuable data references for proteomics studies.
Conflicts of Interest
The authors declare no conflicts of interest.
Open Research
Data Availability Statement
The mass spectrometry raw files and the database search result files have been submitted to the ProteomeXchange Consortium through the iProX partner with ProteomeXchange ID PXD061160.
Supporting Information
| Filename | Description |
|---|---|
| jms5141-sup-0001-Suppl-figures.pdfPDF document, 448.8 KB |
Figure S1. Proteomic analysis results of different quenching methods in four protein sample preparation methods. (A) Venn diagrams show the shared identification peptide numbers in four protein sample preparation methods. (B) Venn diagrams show the shared identification cysteine-containing peptide numbers in four protein sample preparation methods. (C,D) Venn diagrams show the reproducibility for protein (C) and peptide (D) identifications among technical replicates under identical conditions. (E,F) Histograms depict the distribution of protein sequence coverage (E) and the distribution of protein molecular weights (F) in four protein sample preparation methods. To validate the findings, all experiments were repeated three times independently. Figure S2. Analysis of trypsin activity in three quenching conditions under four sample preparation methods. (A) Violin plot demonstrates the statistical analysis of KP/RP digestion rates of three quenching methods in four protein sample preparation methods. (C–F) Histograms illustrate the number of different numbers of missed peptides (0, 1, 2) of three quenching methods in DTT-IAA (C), TCEP-IAA (D), DTT-CAA (E), and TCEP-CAA (F) groups. (G,H) The XIC area comparison of the trypsin self-cleavage peptide “VATVSLPR” across the three quenching methods in two replicates of DTT-IAA group. Three technical replicates were conducted per method. Data are presented as mean ± SEM (Student’s t test, n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure S3. Numbers of alkylation sites and overalkylation sites of four protein sample preparation methods under three conditions: quenching with cysteine, quenching with DTT and no quenching. (A) The number of alkylation sites on cysteine with a localization probability cutoff of 0.75 across the three quenching methods in four protein sample preparation methods. (B–E) The number of alkylation sites on various amino acids with a localization probability cutoff of 0.75 across the three quenching methods in DTT-IAA (B), TCEP-IAA (C), DTT-CAA (D), and TCEP-CAA (E) groups. Technical replicates were performed in triplicate for each group. Data are presented as mean ± SEM (Student’s t test, n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. |
| jms5141-sup-0002-Suppl-tables.xlsxExcel 2007 spreadsheet , 70.5 MB |
Data S1. Supporting Information. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1V. A. Duong and H. Lee, “Bottom-Up Proteomics: Advancements in Sample Preparation,” International Journal of Molecular Sciences 24, no. 6 (2023): 5350.
- 2R. Aebersold and M. Mann, “Mass Spectrometry-Based Proteomics,” Nature 422, no. 6928 (2003): 198–207.
- 3A. Karcini and I. M. Lazar, “The SKBR3 Cell-Membrane Proteome Reveals Telltales of Aberrant Cancer Cell Proliferation and Targets for Precision Medicine Applications,” Scientific Reports 12, no. 1 (2022): 10847.
- 4A. Sebé-Pedrós, M. I. Peña, S. Capella-Gutiérrez, et al., “High-Throughput Proteomics Reveals the Unicellular Roots of Animal Phosphosignaling and Cell Differentiation,” Developmental Cell 39, no. 2 (2016): 186–197.
- 5R. Moaddel, C. Ubaida-Mohien, T. Tanaka, et al., “Proteomics in Aging Research: A Roadmap to Clinical, Translational Research,” Aging Cell 20, no. 4 (2021): e13325.
- 6P. Udomwan, C. Pientong, P. Tongchai, et al., “Proteomics Analysis of Andrographolide-Induced Apoptosis via the Regulation of Tumor Suppressor p53 Proteolysis in Cervical Cancer-Derived Human Papillomavirus 16-Positive Cell Lines,” International Journal of Molecular Sciences 22, no. 13 (2021): 6806.
- 7P. A. Rudnick, K. R. Clauser, L. E. Kilpatrick, et al., “Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in Proteomics Analyses,” Molecular & Cellular Proteomics 9, no. 2 (2010): 225–241.
- 8E. Malmström, O. Kilsgård, S. Hauri, et al., “Large-Scale Inference of Protein Tissue Origin in Gram-Positive Sepsis Plasma Using Quantitative Targeted Proteomics,” Nature Communications 7 (2016): 10261.
- 9D. A. Wolters, M. P. Washburn, and J. R. Yates, 3rd., “An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics,” Analytical Chemistry 73, no. 23 (2001): 5683–5690.
- 10J. R. Yates, 3rd., “Mass Spectral Analysis in Proteomics,” Annual Review of Biophysics and Biomolecular Structure 33 (2004): 297–316.
- 11A. J. Link, J. Eng, D. M. Schieltz, et al., “Direct Analysis of Protein Complexes Using Mass Spectrometry,” Nature Biotechnology 17, no. 7 (1999): 676–682.
- 12W. Choksawangkarn, N. Edwards, Y. Wang, P. Gutierrez, and C. Fenselau, “Comparative Study of Workflows Optimized for In-Gel, In-Solution, and On-Filter Proteolysis in the Analysis of Plasma Membrane Proteins,” Journal of Proteome Research 11, no. 5 (2012): 3030–3034.
- 13B. Woodland, A. Necakov, and J. R. Coorssen, “Optimized Proteome Reduction for Integrative Top-Down Proteomics,” Proteomes 11, no. 1 (2023): 10.
- 14X. Liu, V. Rossio, S. P. Gygi, and J. A. Paulo, “Enriching Cysteine-Containing Peptides Using a Sulfhydryl-Reactive Alkylating Reagent With a Phosphonic Acid Group and Immobilized Metal Affinity Chromatography,” Journal of Proteome Research 22, no. 4 (2023): 1270–1279.
- 15E. B. Getz, M. Xiao, T. Chakrabarty, R. Cooke, and P. R. Selvin, “A Comparison Between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry,” Analytical Biochemistry 273, no. 1 (1999): 73–80.
- 16K. G. Kuznetsova, L. I. Levitsky, M. A. Pyatnitskiy, et al., “Cysteine Alkylation Methods in Shotgun Proteomics and Their Possible Effects on Methionine Residues,” Journal of Proteomics 231 (2021): 104022.
- 17I. V. Turko and S. Sechi, “Acrylamide—A Cysteine Alkylating Reagent for Quantitative Proteomics,” Methods in Molecular Biology 359 (2007): 1–16.
- 18M. L. Nielsen, M. Vermeulen, T. Bonaldi, J. Cox, L. Moroder, and M. Mann, “Iodoacetamide-Induced Artifact Mimics Ubiquitination in Mass Spectrometry,” Nature Methods 5, no. 6 (2008 Jun): 459–460.
- 19E. L. Murphy, A. P. Joy, R. J. Ouellette, and D. A. Barnett, “Optimization of Cysteine Residue Alkylation Using an On-Line LC-MS Strategy: Benefits of Using a Cocktail of Haloacetamide Reagents,” Analytical Biochemistry 619 (2021): 114137.
- 20A. Shevchenko, M. Wilm, O. Vorm, and M. Mann, “Mass Spectrometric Sequencing of Proteins Silver-Stained Polyacrylamide Gels,” Analytical Chemistry 68, no. 5 (1996): 850–858.
- 21P. G. Hains and P. J. Robinson, “The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification,” Journal of Proteome Research 16, no. 9 (2017): 3443–3447.
- 22I. M. Chaiken and E. L. Smith, “Reaction of Chloroacetamide With the Sulfhydryl Group of Papain,” Journal of Biological Chemistry 244, no. 19 (1969): 5087–5094.
- 23Y. Zhang, B. R. Fonslow, B. Shan, M. C. Baek, and J. R. Yates, III, “Protein Analysis by Shotgun/Bottom-Up Proteomics,” Chemical Reviews 113, no. 4 (2013): 2343–2394.
- 24K. C. Ravindra, V. S. Vaidya, Z. Wang, et al., “Tandem Mass Tag-Based Quantitative Proteomic Profiling Identifies Candidate Serum Biomarkers of Drug-Induced Liver Injury in Humans,” Nature Communications 14, no. 1 (2023): 1215.
- 25J. Y. Xu, Y. Xu, Z. Xu, et al., “Protein Acylation Is a General Regulatory Mechanism in Biosynthetic Pathway of Acyl-CoA-Derived Natural Products,” Cell Chemical Biology 25, no. 8 (2018): 984–995.e6.
- 26Y. Qu, X. Wu, A. Anwaier, et al., “Proteogenomic Characterization of MiT Family Translocation Renal Cell Carcinoma,” Nature Communications 13, no. 1 (2022): 7494.
- 27B. Li, F. Guo, H. Hu, et al., “The Characterization of Column Heating Effect in Nanoflow Liquid Chromatography Mass Spectrometry (nanoLC-MS)-Based Proteomics,” Journal of Mass Spectrometry 55, no. 1 (2020): e4441.
- 28Z. Lin, Y. Ren, Z. Shi, et al., “Evaluation and Minimization of Nonspecific Tryptic Cleavages in Proteomic Sample Preparation,” Rapid Communications in Mass Spectrometry 34, no. 10 (2020): e8733.
- 29S. Suttapitugsakul, H. Xiao, J. Smeekens, and R. Wu, “Evaluation and Optimization of Reduction and Alkylation Methods to Maximize Peptide Identification With MS-Based Proteomics,” Molecular BioSystems 13, no. 12 (2017): 2574–2582.
- 30Y. Song, X. Shi, Y. Zou, et al., “Proteome-Wide Identification and Functional Analysis of Ubiquitinated Proteins in Peach Leaves,” Scientific Reports 10, no. 1 (2020): 2447.
- 31L. A. Marotti, Jr., R. Newitt, Y. Wang, R. Aebersold, and H. G. Dohlman, “Direct Identification of a G Protein Ubiquitination Site by Mass Spectrometry,” Biochemistry 41, no. 16 (2002): 5067–5074.
- 32J. Vasilescu, J. C. Smith, D. R. Zweitzig, N. J. Denis, D. S. Haines, and D. Figeys, “Systematic Determination of Ion Score Cutoffs Based on Calculated False Positive Rates: Application for Identifying Ubiquitinated Proteins by Tandem Mass Spectrometry,” Journal of Mass Spectrometry 43, no. 3 (2008): 296–304.
- 33J. R. Wiśniewski, A. Zougman, N. Nagaraj, and M. Mann, “Universal Sample Preparation Method for Proteome Analysis,” Nature Methods 6, no. 5 (2009): 359–362.
- 34C. S. Hughes, S. Moggridge, T. Müller, P. H. Sorensen, G. B. Morin, and J. Krijgsveld, “Single-Pot, Solid-Phase-Enhanced Sample Preparation for Proteomics Experiments,” Nature Protocols 14, no. 1 (2019): 68–85.
- 35H. E. Johnston, K. Yadav, J. M. Kirkpatrick, et al., “Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation Without Magnetic Beads,” Analytical Chemistry 94, no. 29 (2022): 10320–10328.
