LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels.
3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.
Althaus
J S
JS
Neuroscience Therapeutics, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA. john.althaus@wl.com
Schmidt
K R
KR
Fountain
S T
ST
Tseng
M T
MT
Carroll
R T
RT
Galatsis
P
P
Hall
E D
ED
D000818:Animals; D001921:Brain / Q000098:blood supply; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008833:Microcirculation / Q000737:chemistry*; D008858:Microscopy, Phase-Contrast; D009566:Nitrates / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis*
D009566:Nitrates; C039642:peroxynitric acid; C002744:3-nitrotyrosine; D014443:Tyrosine