The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry.
Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched to four different proteins: synaptosomal-associated protein, actin, immunoglobulin alpha Fc receptor, and cGMP-dependent protein kinase 2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility.
Zhan
Xianquan
X
Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 117, Memphis, TN 38163, USA.
Desiderio
Dominic M
DM
D000595:Amino Acid Sequence; D015153:Blotting, Western / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009566:Nitrates / Q000737:chemistry*; D010902:Pituitary Gland / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry*
D009566:Nitrates; D020543:Proteome; D014443:Tyrosine