Liquid- and gas-phase nitration of bovine serum albumin studied by LC-MS and LC-MS/MS using monolithic columns.
Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumin (BSA) and yielded sequence coverages of 95% and 33% with RP-HPLC--ESI--MS and RP-HPLC--ESI--MS/MS, respectively. At least 95% of the tyrosines were covered by the former method, whereas the latter method only detected less than 50% of the tyrosine-containing peptides. Upon liquid-phase nitration of BSA in aqueous solution using an excess of tetranitromethane, at least 16 of the 20 tyrosine residues were found to be nitrated. After exposure of solid BSA samples to gaseous nitrogen dioxide and ozone at atmospherically relevant concentration levels, only 3 nitrated peptides were detected. By use of such a model system, RP-HPLC--ESI--MS proved to be a rapid and highly efficient method for the comprehensive and quantitative detection of protein nitration.
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, A-6020 Innsbruck, Austria.
Wolfgang
W
Walcher
Thomas
T
Franze
Michael G
MG
Weller
Ulrich
U
Pöschl
Christian G
CG
Huber
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009566:Nitrates / Q000737:chemistry*; D009585:Nitrogen Dioxide / Q000378:metabolism; D010126:Ozone / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D009566:Nitrates; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D010126:Ozone; D014357:Trypsin; D009585:Nitrogen Dioxide