LC/MS characterization of undesired products formed during iodoacetamide derivatization of sulfhydryl groups of peptides

Zhihua Yang, Athula B. Attygalle

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

Abstract

Many undesired by-products have been noticed during alkylation with iodoacetamide, a widely used derivatization reaction in proteomics for the determination of sulfhydryl groups in peptides and proteins. We report here that iodoacetamide reacts with the N-terminal NH2 and the C-terminal carboxylic acid groups, in addition to the peripheral residues bearing protic functional groups. If sufficient reaction time is given, the N-terminal NH 2 group is readily dialkylated by iodoacetamide. In fact, the N-terminal NH2 group reacts even faster than the reactive sites present in residues, such as tyrosine or histidine. LC/MS investigations with certain reactive peptides show that by-products are formed in a relatively short reaction time, even at room temperature. Interestingly, derivatives formed in this way are useful for sequence determination of peptides by MS since the intensities of y″ ions are highly suppressed in the spectra of N-terminus mono- and dialkylated peptides, whereas those of b-ions are significantly enhanced. For example, in the spectrum of N,N-dicarboxamidomethyl derivative of Val-Ala-Ala-Phe (VAAF), the y-series ions are virtually absent. On the other hand, when the derivatization takes place at the carboxylic group, the y-series ions are markedly observed in the spectra of these undesired O-carboxamidomethyl derivatives.

Original languageEnglish
Pages (from-to)233-243
Number of pages11
JournalJournal of Mass Spectrometry
Volume42
Issue number2
DOIs
StatePublished - Feb 2007

Keywords

  • Alkylation
  • Iodoacetamide
  • N,N-dicarboxamidomethyl derivatives
  • O-carboxamidomethyl derivatives
  • Proteomics
  • Sulfhydryl group

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