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.