ArticleMolecular & cellular proteomics : MCP2017
Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents.
Article in Molecular & cellular proteomics : MCP, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 77 papers.
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Who cites it
77 citing papers in PubMed, 175 citations in OpenAlex.
- Article
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- Exploring the Potential of Ultrafast Arylation for Capping Cysteine Residues with Fixed Charge Modifications.Journal of the American Society for Mass Spectrometry · 2026Article
- Dual SORT LNPs for multi-organ base editing.Nature biotechnology · 2026Article
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- Quantitative Mapping of NHS Ester-Protein Reactivity Using Native Top-Down Mass Spectrometry.Journal of the American Society for Mass Spectrometry · 2026Article
- Mass Spectrometry-Based Proteomic Analysis of Lysosome-Enriched Fractions.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Comprehensive identification of glutathione peroxidase (GPX) gene family and effect of GhGPX4 on reactive oxygen species metabolism in cotton.Frontiers in plant science · 2026Article
- Single-Cell Nucleus Extraction with Cellular Indexing.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- The Importance of Denaturing Conditions for Protein and Peptide Quantification by Tryptophan Fluorescence.ACS omega · 2025Article
- Sample Preparation for Multi-Omics Analysis: Considerations and Guidance for Identifying the Ideal Workflow.Proteomics · 2025Review
- Analysis of porcine gelatin in gelatin powder and gelatin-containing foods using non-targeted LC-Orbitrap HRMS-based proteomics.Open veterinary journal · 2025Article
- Double alkylation with maleimide-PEG-biotin: An enrichment method for cysteine redox states.Analytical biochemistry · 2025Article
- Detection of Protein Polysulfidation Using a β-(4-Hydroxyphenyl)ethyl Iodoacetamide-Derived Biotin Tag HPB.Analytical chemistry · 2025Article
- Challenges and Insights in Absolute Quantification of Recombinant Therapeutic Antibodies by Mass Spectrometry: An Introductory Review.Antibodies (Basel, Switzerland) · 2025Review
- Proteomic Analysis of Biological Fluids.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Tissue-Specific Biotinylation for Interorgan Communication.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Protein Sample Preparation for Bottom-Up, Label-Free Quantitative Proteomics of Adipose Tissue.Methods in molecular biology (Clifton, N.J.) · 2025Article
17 more citing papers are in PubMed but not listed here.
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
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Abstract
Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.
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