AminoxyTMT : A novel multi-functional reagent for characterization of protein carbonylation
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...
| Publié dans: | BioTechniques. - 1991. - 60(2016), 4 vom: 14. Apr., Seite 186-8, 190, 192-6 |
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| Auteur principal: | |
| Autres auteurs: | , , , |
| Format: | Article en ligne |
| Langue: | English |
| Publié: |
2016
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| Accès à la collection: | BioTechniques |
| Sujets: | Journal Article Research Support, N.I.H., Extramural AminoxyTMT Immunoblotting Mass spectrometry Post translational modification Protein carbonylation Tandem Mass Tag Indicators and Reagents Peptides |
| Résumé: | 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 |
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| Description: | Date Completed 28.12.2016 Date Revised 30.12.2016 published: Electronic-eCollection Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/000114402 |