title: AminoxyTMT: A novel multi-functional reagent for characterization of protein carbonylation.
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.
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN.|Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN.
authors.forename: Somaieh
authors.initials: S
authors.lastname: Afiuni-Zadeh
authors.affiliation: Thermo Fisher Scientific, Rockford, IL.
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
authors.affiliation: Thermo Fisher Scientific, Rockford, IL.
authors.forename: Sergei I
authors.initials: SI
authors.lastname: Snovida
authors.affiliation: Thermo Fisher Scientific, Rockford, IL.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Bomgarden
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007202:Indicators and Reagents / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D010455:Peptides / Q000737:chemistry; D050050:Protein Carbonylation; D011506:Proteins / Q000737:chemistry*
chemical_list: D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins
keywords: AminoxyTMT; Immunoblotting; Mass spectrometry; Post translational modification; Protein carbonylation; Tandem Mass Tag