title: Identification of nitrotyrosine containing peptides using combined fractional diagonal chromatography (COFRADIC) and off-line nano-LC-MALDI.
abstract: Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Trine R
authors.initials: TR
authors.lastname: Larsen
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bache
authors.forename: Jan Bert
authors.initials: JB
authors.lastname: Gramsbergen
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D015538:Nitrosation; D010446:Peptide Fragments / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine; D045304:Cytochromes c