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Selective solid‐phase isolation of methionine‐containing peptides and subsequent matrix‐assisted laser desorption/ionisation mass spectrometric detection of methionine‐ and of methionine‐sulfoxide‐containing peptides
Author(s) -
Grunert Tom,
Pock Katharina,
Buchacher Andrea,
Allmaier Günter
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1110
Subject(s) - methionine sulfoxide , chemistry , methionine , matrix assisted laser desorption/ionization , sulfoxide , mass spectrometry , chromatography , amino acid , biochemistry , organic chemistry , desorption , adsorption
Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine‐ and methionine‐sulfoxide‐containing peptides, involving application of methionine‐specific solid‐phase reagent chemistry combined with matrix‐assisted laser desorption/ionisation mass spectrometry (MALDI‐MS). In the first step, methionine‐containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine‐sulfoxide‐containing peptides and other methionine‐free peptides are not bound and are washed out. The wash solution is used for MALDI‐MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post‐source decay (PSD) fragment ion analysis. Methionine‐sulfoxide‐containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2‐mercaptoethanol at pH 8.5–8.8. The resulting peptides, mainly methionine‐containing peptides, are analysed in a straightforward manner by MALDI‐MS and seamless PSD. The strategy allows the fast identification of methionine‐ and methionine‐sulfoxide‐containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine‐containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine‐free peptides can be quite strongly bound non‐covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that oxidation of some methionine residues occurred to a minor extent during the solid‐phase isolation steps. Copyright © 2003 John Wiley & Sons, Ltd.