Chemical labeling and enrichment of nitrotyrosine-containing peptides.
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 peptides 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 II in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics.
Abello
Nicolas
N
Department of Pharmacy, Analytical Biochemistry, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Barroso
Begona
B
Kerstjens
Huib A M
HA
Postma
Dirkje S
DS
Bischoff
Rainer
R
D000818:Animals; D002417:Cattle; D013058:Mass Spectrometry; D009566:Nitrates / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
D009566:Nitrates; D010446:Peptide Fragments; D010455:Peptides; C002744:3-nitrotyrosine; D014443:Tyrosine