Cysteine alkylation methods in shotgun proteomics and their possible effects on methionine residues

Kuznetsova, Ksenia G.; Levitsky, Lev I.; Pyatnitskiy, Mikhail A.; Ilina, Irina Y.; Bubis, Julia A.; Solovyeva, Elizaveta M.; Zgoda, Victor G.; Gorshkov, Mikhail V.; Moshkovskii, Sergei A.

doi:10.1016/j.jprot.2020.104022

Cited by 19 publications

(15 citation statements)

References 41 publications

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“…Criteria seemingly vary from lab-to-lab (and journal-to-journal). Thus, substantial sequence coverage—some argue, with solid rationale, that only 100% coverage is optimal [ 5 ] or at least several peptides that roughly cover the full span of the sequence (i.e., minimally near the C and D termini and the middle)—is required to confidently assume a canonical protein identification [ 28 , 80 ]. We suggest using a minimum of three peptides, that span the range of the apparent canonical protein sequence, as criteria for a positive identification: this is further strengthened if proteoforms have first been resolved (i.e., top-down analysis) [ 27 , 28 , 81 ].…”

Section: Discovery Proteomicsmentioning

confidence: 99%

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Proteomes Are of Proteoforms: Embracing the Complexity

2021

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Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplifying their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. While having a catalogue of amino acid sequences provides invaluable information, this is the Proteome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we must assess the myriad of proteoforms that arise at any point before, after, and between translation and transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as well as newly defined species. There are numerous analytical methods currently used to address proteome depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We thus discuss both pros and cons of available approaches and where improvements or refinements are needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest that advances lie in transdisciplinarity via integration of current proteomic methods to yield a unified discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolutionary) shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptomics. We must embrace the complexity. Yes, these are the hard questions, and this will not be easy…but where is the fun in easy?

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Section: Discovery Proteomicsmentioning

confidence: 99%

“…Thus, one of the main complications that arises is a lack of standardized methods and thus poor reproducibility between different laboratories [ 80 , 83 ]. Regrettably, this has likely been a problem since the first SDS-PAGE gel was resolved in the second lab to ever try the method.…”

Section: Discovery Proteomicsmentioning

confidence: 99%

Proteomes Are of Proteoforms: Embracing the Complexity

2021

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“…Using other alkylating agents (e.g., iodoacetamide, iodoacetic acid, 4-vinylpiridine, and acrylamide) to block free cysteine residues at the initial steps of the protocol was not evaluated here, but they could also be useful. Potential side reactions related to the presence of Cys-blocking groups should definitively be explored in depth to develop a well-characterized protocol [52][53][54][55][56].…”

Section: Artificial Modifications Introduced During Sample Processing By the In-solution Bfd Protocolmentioning

confidence: 99%

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

et al. 2021

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“…However, later work by Hains [42] showed that although CAM had a higher specificity compared to IAM, its use resulted in significant oxidation of methionine (M) and tryptophan (W) residues. Recent results comparing alkylating agents such as IAM, CAM, IAA, 4-VP, and MMTS showed that CAM is the preferred alkylating agent for sample preparation [67]. Alternatively, a smaller amount of IAM at a concentration of 14 mM [63] can be used for alkylation instead of the commonly used amount of 50-55 mM [4,68].…”

Section: 13mentioning

confidence: 99%

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

2021

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Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is now the main analytical method for the identification and quantification of peptides and proteins in biological samples. In modern research, identification of biomarkers and their quantitative comparison between samples are becoming increasingly important for discovery, validation, and monitoring. Such data can be obtained following specific signals after fragmentation of peptides using multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods, with high specificity, accuracy, and reproducibility. In addition, these methods allow measurement of the amount of posttranslationally modified forms and isoforms of proteins. This review article describes the basic principles of MRM assays, guidelines for sample preparation, recent advanced MRM-based strategies, applications and illustrative perspectives of MRM/PRM methods in clinical research and molecular biology.

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Cysteine alkylation methods in shotgun proteomics and their possible effects on methionine residues

Cited by 19 publications

References 41 publications

Proteomes Are of Proteoforms: Embracing the Complexity

Proteomes Are of Proteoforms: Embracing the Complexity

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

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