ArticleMolecular & cellular proteomics : MCP2014
Facilitating protein disulfide mapping by a combination of pepsin digestion, electron transfer higher energy dissociation (EThcD), and a dedicated search algorithm SlinkS.
Article in Molecular & cellular proteomics : MCP, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 39 papers.
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39 citing papers in PubMed, 86 citations in OpenAlex.
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- Structural characterization of intra- and intermolecular disulfide bonds in voltage-dependent anion channel 3 (VDAC3) protein from Rattus norvegicus by high-resolution mass spectrometry.Analytical and bioanalytical chemistry · 2025Article
- Detailed Structural Elucidation of Antibody-Drug Conjugate Biotransformation Species Using High Resolution Multiple Reaction Monitoring Mass Spectrometry with Orthogonal Dissociation Methods.ACS pharmacology & translational science · 2025Article
- Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry.ACS measurement science au · 2024Review
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- Strategies for mass spectrometry-based phosphoproteomics using isobaric tagging.Expert review of proteomics · 2021Review
- Advances in quantitative high-throughput phosphoproteomics with sample multiplexing.Proteomics · 2021Review
- ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping.Molecular & cellular proteomics : MCP · 2020Article
- Top-Down Characterization of an Intact Monoclonal Antibody Using Activated Ion Electron Transfer Dissociation.Analytical chemistry · 2020Article
- Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications.Chemical reviews · 2020Review
- Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.Analytical chemistry · 2020Article
- Computational methods in mass spectrometry-based structural proteomics for studying protein structure, dynamics, and interactions.Computational and structural biotechnology journal · 2020Review
- Top-Down Analysis of Disulfide-Linked Proteins Using Photoinduced Radical Reactions and ET-DDC.International journal of mass spectrometry · 2019Article
- Analysis of protein chlorination by mass spectrometry.Redox biology · 2019Article
- Experimental Assignment of Disulfide-Bonds in Purified Proteins.Current protocols in protein science · 2019Article
- Comprehensive identification of protein disulfide bonds with pepsin/trypsin digestion, Orbitrap HCD and Spectrum Identification Machine.Journal of proteomics · 2019Article
- Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD.The Analyst · 2018Article
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3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biological functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample preparation and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissociation (EThcD). In EThcD fragmentation of disulfide-bridged peptides, electron transfer dissociation preferentially leads to the cleavage of the S-S bonds, generating two intense disulfide-cleaved peptides as primary fragment ions. Subsequently, higher energy collision dissociation primarily targets unreacted and charge-reduced precursor ions, inducing peptide backbone fragmentation. SlinkS is able to provide the accurate monoisotopic precursor masses of the two disulfide-cleaved peptides and the sequence of each linked peptide by matching the remaining EThcD product ions against a linear peptide database. The workflow was validated using a protein mixture containing six proteins rich in natural disulfide bridges. Using this pepsin-based workflow, we were able to efficiently and confidently identify a total of 31 unique Cys-Cys bonds (out of 43 disulfide bridges present), with no disulfide reshuffling products detected. Pepsin digestion not only outperformed trypsin digestion in terms of the number of detected authentic Cys-Cys bonds, but, more important, prevented the formation of artificially reshuffled disulfide bridges due to protein digestion under neutral pH. Our new workflow therefore provides a precise and generic approach for disulfide bridge mapping, which can be used to study protein folding, structure, and stability.
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