title: Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method.
abstract: In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO(2) for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23.
authors.affiliation: Centre for Molecular and Biomedical Sciences, Faculty of Health and Life Sciences, Coventry University, Coventry CV1 5FB, UK.
authors.forename: Dominic
authors.initials: D
authors.lastname: Matters
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Cooper
authors.forename: Liam
authors.initials: L
authors.lastname: McDonnell
authors.forename: Jesus
authors.initials: J
authors.lastname: Iniesta
authors.forename: John
authors.initials: J
authors.lastname: Heptinstall
authors.forename: Peter
authors.initials: P
authors.lastname: Derrick
authors.forename: David
authors.initials: D
authors.lastname: Walton
authors.forename: Ian
authors.initials: I
authors.lastname: Peterson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D017357:Cyclotrons; D004529:Egg White; D004563:Electrochemistry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D009566:Nitrates; D014443:Tyrosine; C421864:hen egg lysozyme; D009113:Muramidase