Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis

Z. Darula, Katalin F. Medzihradszky

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Iodoacetamide is perhaps the most widely used reagent for the alkylation of free sulfhydryls in proteomic experiments. Here, we report that both incomplete derivatization of Cys side chains and overalkylation of the peptides may lead to the misassignment of glycoforms when LC-MS/MS with electron-transfer dissociation (ETD) alone is used for the structural characterization of glycopeptides. Accurate mass measurements do not help, because the elemental compositions of the misidentified and correct modifications are identical. Incorporation of higher-energy C-trap dissociation (HCD), i.e., beam-type collision-induced dissociation data into the database searches with ETD data may prove decisive in most cases. However, the carbamidomethylation of Met residues leads to sulfonium ether formation, and the resulting fixed positive charge triggers a characteristic fragmentation, that eliminates the normal Y1 fragment from the HCD spectra of N-linked glycopeptides, producing an abundant Y1-48 Da ion instead (the nominal mass difference is given relative to the unmodified amino acid sequence), that easily can be mistaken for the side chain loss from Met sulfoxide. In such cases, good quality ETD data may indicate the discrepancy, and will also display abundant fragments due to CH3-S-CH2CONH2 elimination from the charge-reduced precursor ions. Our observations also draw attention to the underreported interference of different unanticipated covalent modifications.

Original languageEnglish
Pages (from-to)6297-6302
Number of pages6
JournalAnalytical Chemistry
Volume87
Issue number12
DOIs
Publication statusPublished - Jun 16 2015

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Glycopeptides
Polysaccharides
sulfoxide
Electrons
Ions
Iodoacetamide
Alkylation
Ether
Amino Acids
Peptides
Chemical analysis
Experiments

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis. / Darula, Z.; Medzihradszky, Katalin F.

In: Analytical Chemistry, Vol. 87, No. 12, 16.06.2015, p. 6297-6302.

Research output: Contribution to journalArticle

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