Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

Paulech, Jana; Solis, Nestor; Cordwell, Stuart J.

doi:10.1016/j.bbapap.2012.08.002

Cited by 54 publications

(53 citation statements)

References 35 publications

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“…To couple this method to site determination in tissues that are not treated with exogenous oxidants, care should be taken to avoid formation of artifacts by performing extractions at low pH, in solutions containing strong denaturants (e.g. detergents), low pH alkylating agents (46), and metal ion chelators. Protease inhibition is of high importance, as the method is particularly sensitive to nontryptic proteolysis occurring at any stage in the protocol.…”

Section: Fig 3 Schematic Of Scx-hilic Methods For the Enrichment Of mentioning

confidence: 99%

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Paulech

Liddy

Engholm-Keller

et al. 2015

Molecular & Cellular Proteomics

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Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO 2 H] and sulfonic [Cys-SO 3 H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO 2 H/SO 3 H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pK a Cys-SO 3 H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO 2 H/SO 3 H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO 2 H/SO 3 H (up to 80%) from other modified peptides. We identified 181 Cys-SO 2 H/SO 3 H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H 2 O 2 (<100 M) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion.

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Section: Fig 3 Schematic Of Scx-hilic Methods For the Enrichment Of mentioning

confidence: 99%

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Paulech

Liddy

Engholm-Keller

et al. 2015

Molecular & Cellular Proteomics

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“…46 The following compounds have normally been employed for alkylation: iodoacetamide (IAA), acrylamide (AA), N-ethylmaleimide (N-EM), and 4-vinylpyridine (4-VP). 41, 47–50 …”

Section: Introductionmentioning

confidence: 99%

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

et al. 2017

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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, the reaction incompletion and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with the 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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“…The principal issue is that the thiol-blocking agents commonly used in BST methods display reactivity with cysteine S-sulfenic acid, which is a key intermediate in thiol oxidation (54,55). Uncertainty regarding the species labeled by thiol-blocking agents introduces ambiguity in interpretation.…”

Section: The Expanding Landscape Of the Thiol Redox Proteomementioning

confidence: 99%

The Expanding Landscape of the Thiol Redox Proteome

Yang

Carroll

Liebler

2016

Molecular & Cellular Proteomics

182

111

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Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO 2 H), S-sulfonylation (-SO 3 H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes.

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Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

Cited by 54 publications

References 35 publications

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

The Expanding Landscape of the Thiol Redox Proteome

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