دورية أكاديمية
Identification by MS/MS of disulfides produced by a functional redox transition.
| العنوان: | Identification by MS/MS of disulfides produced by a functional redox transition. |
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| المؤلفون: | Mauri P; Institute for Biomedical Technologies, National Research Council, Viale Fratelli Cervi, Segrate-Milano, Italy., Toppo S, De Palma A, Benazzi L, Maiorino M, Ursini F |
| المصدر: | Methods in enzymology [Methods Enzymol] 2010; Vol. 473, pp. 217-25. |
| نوع المنشور: | Journal Article; Review |
| اللغة: | English |
| بيانات الدورية: | Publisher: Academic Press Country of Publication: United States NLM ID: 0212271 Publication Model: Print Cited Medium: Internet ISSN: 1557-7988 (Electronic) Linking ISSN: 00766879 NLM ISO Abbreviation: Methods Enzymol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: New York, Academic Press. |
| مواضيع طبية MeSH: | Disulfides/*analysis , Disulfides/*metabolism , Tandem Mass Spectrometry/*methods, Animals ; Chromatography, Liquid/methods ; Data Interpretation, Statistical ; Humans ; Oxidation-Reduction ; Spectrometry, Mass, Electrospray Ionization/methods |
| مستخلص: | Among posttranslational modifications of proteins entailed with signal transduction, the redox transition is today brought to the focus as a major biochemical event accounting for the signaling functions of reactive oxygen species. Thermodynamic and kinetic criteria highlight hydroperoxides and protein disulfides as signaling and transducer elements, respectively, and growing biochemical evidence supports this notion. The protein Cys residue involved in this function must react fast and specifically with the oxidant and then with a second accessible Cys yielding the disulfide. These kinetic and structural constraints are shared with peroxidases and peroxiredoxins, which are competitors for the signaling hydroperoxide. In this chapter, a procedure based on MS/MS analysis for inter- and intrachain disulfide assignment in proteins undergoing redox-switch is presented. While the sensitivity of the modern MS/MS instruments permits the sequencing of double peptides linked by a disulfide bond, the major pitfall of the proteomic procedure is the thiol-disulfide scrambling taking place at the alkaline pH needed for the proteolytic reaction of trypsin. Instead, the use of pepsin at acidic pH prevents the disulfide scrambling, but the specificity of the proteolytic reaction is low and thus the complexity of fragmentation increases. We succeeded to limit this problem by heuristically assuming a conserved pepsin cleavage pattern of the protein both in the oxidized and the reduced form. Asymmetric cleavage of the disulfide by collisional fragmentation further corroborated the identification. In conclusion, the use of pepsin, integrated by a minimal computation, appears suitable for positively assigning inter- and intrachain disulfides generated by a functional redox-switch. (Copyright (c) 2010 Elsevier Inc. All rights reserved.) |
| Number of References: | 17 |
| المشرفين على المادة: | 0 (Disulfides) |
| تواريخ الأحداث: | Date Created: 20100602 Date Completed: 20100902 Latest Revision: 20100601 |
| رمز التحديث: | 20221213 |
| DOI: | 10.1016/S0076-6879(10)73011-1 |
| PMID: | 20513480 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1557-7988 |
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| DOI: | 10.1016/S0076-6879(10)73011-1 |