In vitro and in vivo protein-bound tyrosine nitration characterized by diagonal chromatography.
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.
Ghesquière
Bart
B
Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium.
Colaert
Niklaas
N
Helsens
Kenny
K
Dejager
Lien
L
Vanhaute
Caroline
C
Verleysen
Katleen
K
Kas
Koen
K
Timmerman
Evy
E
Goethals
Marc
M
Libert
Claude
C
Vandekerckhove
Joël
J
Gevaert
Kris
K
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
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