AminoxyTMT: A novel multi-functional reagent for characterization of protein carbonylation

Somaieh Afiuni-Zadeh, John C. Rogers, Sergei I. Snovida, Ryan D. Bomgarden, Timothy J. Griffin

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Protein carbonylation is a common oxidative stress (OS)–driven post-translational modification (PTM). Proteome- wide carbonylation events can best be characterized using a combination of analytical approaches. Immunoblotting of carbonylated proteins provides data on the extent of modifications within complex samples, as well as a broad comparison of carbonylation profiles between different biological states (e.g., disease versus control), while mass spectrometry (MS)–based analysis provides information on proteins susceptible to carbonylation, as well as the potential for quantitative characterization of specific sites of amino acid modification. Here, we present a novel use for aminoxyTMT, a derivative of the Tandem Mass Tag (TMT) isobaric labeling reagent, which utilizes an aminooxy functional group for covalent labeling of reactive carbonyls in proteins. When coupled with anti-TMT antibody, we demonstrate the use of aminoxyTMT for immunoblot profiling of protein carbonylation in complex mixtures, as well as enrichment of modified peptides from these mixtures. Proof-of-principle experiments also show the amenability of aminoxyTMT-labeled carbonylated peptides enriched from complex mixtures to identification using tandem MS (MS/MS) and database searching, as well as quantitative analysis using TMT-based reporter ion intensity measurements.

Original languageEnglish (US)
Pages (from-to)186-196
Number of pages11
JournalBioTechniques
Volume60
Issue number4
DOIs
StatePublished - Apr 2016

Bibliographical note

Publisher Copyright:
© 2016, Eaton Publishing Company. All Rights Reserved.

Keywords

  • AminoxyTMT
  • Immunoblotting
  • Mass spectrometry
  • Post translational modification
  • Protein carbonylation
  • Tandem mass tag

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