Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry.
Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.
Lee
Soo Jae
SJ
Institute of Biomedical Science and Technology, Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Korea.
Lee
Jung Rok
JR
Kim
Young Hwan
YH
Park
Yoon Shin
YS
Park
Sang Ick
SI
Park
Hyung Soon
HS
Kim
Kwang Pyo
KP
D000804:Angiotensin II / Q000737:chemistry*; D009566:Nitrates / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry*
D009566:Nitrates; D000804:Angiotensin II; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D009584:Nitrogen