Model studies on iTRAQ modification of peptides

Sequence-dependent reaction specificity

John E. Wiktorowicz, Robert D. English, Zheng Wu, Alexander Kurosky

    Research output: Contribution to journalArticle

    9 Citations (Scopus)

    Abstract

    A multiplexed peptide quantification strategy using the iTRAQ reagent has been described for relative measurements of peptides in digested protein mixtures. To validate the chemical specificity of the iTRAQ reaction, we have performed a detailed study of iTRAQ reactivity with two sets of synthetic peptides. The first set of peptides had sequences of Tyr-Xaa-Ser-Glu-Gly-Leu- Ser-Lys and Tyr-Xaa-Ser-Glu-Tyr-Leu-Ser-Lys where Xaa = Ala, Pro, Trp, Tyr, or Glu and was designed to study the extent of O-acylation by iTRAQ, especially hydroxyl-containing residues in different positions. The second set of peptides included Ala-Ser-Glu-His-Ala-Xaa-Tyr-Gly where Xaa = Ser, Thr, or Tyr and was selected to investigate the effect of histidyl residues separated by one amino acid residue from seryl, tyrosyl, or threonyl residues. Our findings indicated that, in addition to variable levels of O-acylation of nonsequence-specific hydroxyl-containing residues, significant sequence-specific O-acylation of seryl, threonyl, and tyrosyl hydroxyls occurred when separated one residue removed from a histidyl residue, that is, (Tyr/Ser)-Xaa-His or His-Xaa-(Tyr/Ser/Thr). This behavior was verified by a separate spiking experiment of one of the first set of peptides into Escherichia coli protein extracts, followed by retention time targeted LC-MS/MS to demonstrate the occurrence of modifications in a complex mixture. These sequence-dependent O-acylation modifications can be confounding factors to accurate MS quantification. Reversal of peptide O-acylation by the iTRAQ reagent can be accomplished by reaction with hydroxylamine with virtually no cleavage of N-acylation and is a recommended modification of the iTRAQ protocol for many applications.

    Original languageEnglish (US)
    Pages (from-to)1512-1520
    Number of pages9
    JournalJournal of Proteome Research
    Volume11
    Issue number3
    DOIs
    StatePublished - Mar 2 2012

    Fingerprint

    Acylation
    Peptides
    Hydroxyl Radical
    Hydroxylamine
    Escherichia coli Proteins
    Complex Mixtures
    Amino Acids
    Proteins

    Keywords

    • hydroxyl amino acid reactivity
    • iTRAQ
    • mass spectrometry
    • peptide O-acylation
    • quantitative proteomics
    • sequence-specific modification

    ASJC Scopus subject areas

    • Biochemistry
    • Chemistry(all)

    Cite this

    Model studies on iTRAQ modification of peptides : Sequence-dependent reaction specificity. / Wiktorowicz, John E.; English, Robert D.; Wu, Zheng; Kurosky, Alexander.

    In: Journal of Proteome Research, Vol. 11, No. 3, 02.03.2012, p. 1512-1520.

    Research output: Contribution to journalArticle

    Wiktorowicz, John E. ; English, Robert D. ; Wu, Zheng ; Kurosky, Alexander. / Model studies on iTRAQ modification of peptides : Sequence-dependent reaction specificity. In: Journal of Proteome Research. 2012 ; Vol. 11, No. 3. pp. 1512-1520.
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