期刊
TALANTA
卷 80, 期 4, 页码 1503-1512出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2009.02.002
关键词
Nitrotyrosine; Tyrosine nitration; NHS-acetate; NHS-biotin; Mass spectrometry; Peroxynitrite
资金
- University of Groningen
- The Netherlands Proteomics Center (NPC)
Protein tyrosine nitration (PTN) is a post-translational modification of proteins associated with a number of inflammatory diseases. While PTN is rather selective (not all proteins are modified and within a protein, only certain tyrosines are subject to nitration), no consensus sequence has been identified. Since PTN is a low-abundance post-translational modification, it is necessary to enrich modified proteins and/or to detect them with high selectivity and sensitivity. Until now this has been mostly accomplished with anti-nitrotyrosine antibodies in combination with two-dimensional gel electrophoresis and mass spectrometry. We propose a chemical labeling approach designed to allow enrichment of tyrosine-nitrated peptides independent of the sequence context, which is a potential shortcoming of antibody-based approaches. In this procedure, all amines are blocked by acetylation followed by conversion of nitrotyrosine to aminotyrosine and biotinylation of aminotyrosine. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes thereby reducing sample loss. Free biotin is subsequently removed with a strong cation exchanger, the labeled pepticles are enriched on an immobilized avidin column and the enriched peptides analyzed by LC-MS/MS. As a proof of concept, this method was successfully applied to the enrichment of tyrosine-nitrated angiotensin 11 in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics. (C) 2009 Elsevier B.V. All rights reserved.
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