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 HPL...

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Bibliographic Details
Published in:Molecular & cellular proteomics 2009-12, Vol.8 (12), p.2642-2652
Main Authors: Ghesquière, Bart, Colaert, Niklaas, Helsens, Kenny, Dejager, Lien, Vanhaute, Caroline, Verleysen, Katleen, Kas, Koen, Timmerman, Evy, Goethals, Marc, Libert, Claude, Vandekerckhove, Joël, Gevaert, Kris
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Blood Proteins - metabolism
Cattle
Cell Extracts
Chromatography, High Pressure Liquid - methods
Chromatography, Reverse-Phase
Disease Models, Animal
Humans
Jurkat Cells
Mice
Mice, Inbred C57BL
Molecular Sequence Data
Oxidation-Reduction
Peptides - chemistry
Peptides - isolation & purification
Proteome - metabolism
Proteomics - methods
Salmonella - physiology
Serum Albumin, Bovine - chemistry
Serum Albumin, Bovine - metabolism
Shock, Septic - blood
Shock, Septic - microbiology
Tetranitromethane - metabolism
Thiosulfates - metabolism
Tyrosine - analogs & derivatives
Tyrosine - metabolism
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Summary: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.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M900259-MCP200