ArticleMolecular bioSystems2017
Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.
Article in Molecular bioSystems, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 44 papers.
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Who cites it
44 citing papers in PubMed.
- Canine Tear Proteomics: A New Frontier in Veterinary Ophthalmology.Veterinary ophthalmology · 2026Review
- Mass Spectrometry-Based Peptidomics for the Discovery and Profiling of Endogenous Peptides in Crustacean Hemolymph.ACS omega · 2026Article
- Exploring the Potential of Ultrafast Arylation for Capping Cysteine Residues with Fixed Charge Modifications.Journal of the American Society for Mass Spectrometry · 2026Article
- Protein Extraction Protocol for Legumes Compatible with Detergent: Gel Fractionation and Mass Spectrometry.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Sample Preparation for Multi-Omics Analysis: Considerations and Guidance for Identifying the Ideal Workflow.Proteomics · 2025Review
- Assessing Substrate Scope of the Cyclodehydratase LynD by mRNA Display-Enabled Machine Learning Models.Biochemistry · 2025Article
- Design of experiments for the optimization of sample preparation for bottom-up targeted protein LC-MS/MS workflows.Bioanalysis · 2025Article
- Multiplexed phosphoproteomics of low cell numbers using SPARCE.Communications biology · 2025Article
- Challenges and Insights in Absolute Quantification of Recombinant Therapeutic Antibodies by Mass Spectrometry: An Introductory Review.Antibodies (Basel, Switzerland) · 2025Review
- Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics.Nature communications · 2024Article
- Peptide and Protein Cysteine Modification Enabled by Hydrosulfuration of Ynamide.ACS central science · 2024Article
- Design and validation of a method for evaluating medical device cleanliness by recovering and quantifying residual proteins on stainless plates.Scientific reports · 2024Article
- Reply: Does the polyubiquitination pathway operate inside intact chloroplasts to remove proteins?The Plant cell · 2024Article
- Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry.ACS measurement science au · 2024Review
- K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors.Life science alliance · 2024Article
- Changes in the proteome of Apis mellifera acutely exposed to sublethal dosage of glyphosate and imidacloprid.Environmental science and pollution research international · 2024Article
- Methionine Alkylation as an Approach to Quantify Methionine Oxidation Using Mass Spectrometry.Journal of the American Society for Mass Spectrometry · 2024Article
- Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation.Nature chemistry · 2024Article
- Biophysical Analysis of Therapeutic Antibodies in the Early Development Pipeline.Biologics : targets & therapy · 2024Review
- Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes.Advances in experimental medicine and biology · 2024Article
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4 authors.
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Abstract
Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, incomplete reactions and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, thiopropyl-sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.
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