title: Quantification of nitrated tryptophan in proteins and tissues by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry.
abstract: Aromatic amino acids are targets of reactive nitrogen species (RNS) such as peroxynitrite (ONOO(-)) and nitrogen dioxide. It is known that tryptophan (Trp) as well as tyrosine is nitrated, generated isomers. However, no quantitative method to determine nitrotryptophan (NO(2)Trp) in proteins has been developed so far. In this study, we have developed a method for the quantification of Trp and NO(2)Trp isomers, 2-, 4- and 6-NO(2)Trp, which uses liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In order to confirm the applicability of our method to in vitro and in vivo system, we measured protein-bound NO(2)Trp levels in ONOO(-) treated bovine serum albumin (BSA) and in liver of B6C3F1 mice at 2, 4, and 8h after administration of 300 mg/kg acetaminophen (APAP). A mass spectrometer equipped with an electrospray ionization source using a crossflow counter electrode and ran in the positive ion mode (ESI(+)) was used for multiple reaction monitoring (MRM) of transitions 205-->188, 250-->130, 250-->159 and 250-->233 for Trp, 2-, 4- and 6-NO(2)Trp, respectively. The recoveries from mice liver samples were 98.3-105.9% for each compound. The limits of quantification were 50, 3.0, 10 and 4.0 nM for Trp, 2-, 4- and 6-NO(2)Trp, respectively. In in vitro experiments demonstrated that all isomers of NO(2)Trp were detectable from BSA treated with ONOO(-) and the amount generated decreased in the order of 6-, 4- and 2-NO(2)Trp. In in vivo experiments, 4- and 6-NO(2)Trp were detected in the liver of mice administered APAP. The concentration range of 4- and 6-NO(2)Trp per mol of Trp in the sample was 2.24-3.92 and 26.96-32.71 nmol/mol of Trp, and its existence in vivo was confirmed for the first time with our method. The LC-ESI-MS/MS method was able to determine protein-bound NO(2)Trp in a small amount of tissue sample, and is therefore applicable not only as a biomarker of RNS, but also as a mean to clarify novel mechanisms underlying RNS-related tissue damage.
authors.affiliation: Department of Analytical Chemistry, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ishii
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ogara
authors.forename: Tsunenobu
authors.initials: T
authors.lastname: Katsumata
authors.forename: Takashi
authors.initials: T
authors.lastname: Umemura
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nishikawa
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Iwasaki
authors.forename: Rie
authors.initials: R
authors.lastname: Ito
authors.forename: Koichi
authors.initials: K
authors.lastname: Saito
authors.forename: Masao
authors.initials: M
authors.lastname: Hirose
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakazawa
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D018712:Analgesics, Non-Narcotic / Q000633:toxicity; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007536:Isomerism; D008099:Liver / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D015394:Molecular Structure; D009566:Nitrates / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis*; D013047:Specific Pathogen-Free Organisms; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014364:Tryptophan / Q000032:analysis*
chemical_list: D018712:Analgesics, Non-Narcotic; D009566:Nitrates; D011506:Proteins; D030421:Peroxynitrous Acid; D012710:Serum Albumin, Bovine; D000082:Acetaminophen; D014364:Tryptophan