Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteins

Abstract

The nitric oxide-mediated Tyr-nitration of endogenous proteins is associated with several pathological and physiological processes. In order to investigate the presence – and potential roles – of Tyr-nitration in the human pituitary, a large-format two-dimensional gel separation plus a Western blot against a specific anti-3-nitrotyrosine antibody were used to separate and detect nitroproteins from a human pituitary proteome. The nitroproteins were subjected to in-gel trypsin digestion, and high-sensitivity vacuum matrix-assisted laser desorption/ionization (vMALDI) linear ion-trap tandem mass spectrometry was used to analyze the tryptic peptides. Those MS/MS data were used to determine the amino acid sequence and the specific nitration site of each tryptic nitropeptide, and were matched to corresponding proteins with Bioworks TuboSEQUEST software. Compared to our previous study, 16 new nitrotyrosine-immunoreactive positive Western blot spots were found within the area pI 3.0–10 and M r 10–100 kDa. Four new nitroproteins were discovered: the stanniocalcin 1 precursor—involved in calcium and phosphate metabolism; mitochondrial co-chaperone protein HscB, which might act as a co-chaperone in iron–sulfur cluster assembly in mitochrondria; progestin and adipoQ receptor family member III—a seven-transmembrane receptor; proteasome subunit alpha type 2—involved in an ATP/ubiquitin-dependent non-lysosomal proteolytic pathway. Those data demonstrate that nitric oxide-mediated Tyr-nitration might participate in various biochemical, metabolic, and pathological processes in the human pituitary.