title: In vitro and in vivo protein-bound tyrosine nitration characterized by diagonal chromatography.
abstract: A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.
authors.affiliation: Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Ghesquière
authors.forename: Niklaas
authors.initials: N
authors.lastname: Colaert
authors.forename: Kenny
authors.initials: K
authors.lastname: Helsens
authors.forename: Lien
authors.initials: L
authors.lastname: Dejager
authors.forename: Caroline
authors.initials: C
authors.lastname: Vanhaute
authors.forename: Katleen
authors.initials: K
authors.lastname: Verleysen
authors.forename: Koen
authors.initials: K
authors.lastname: Kas
authors.forename: Evy
authors.initials: E
authors.lastname: Timmerman
authors.forename: Marc
authors.initials: M
authors.lastname: Goethals
authors.forename: Claude
authors.initials: C
authors.lastname: Libert
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002417:Cattle; D002457:Cell Extracts; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D004195:Disease Models, Animal; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012475:Salmonella / Q000502:physiology; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D012772:Shock, Septic / Q000097:blood / Q000382:microbiology; D013774:Tetranitromethane / Q000378:metabolism; D013885:Thiosulfates / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D002457:Cell Extracts; D010455:Peptides; D020543:Proteome; D013885:Thiosulfates; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine; C017717:sodium thiosulfate; D013774:Tetranitromethane