Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometry

Aaron Sarver, N. Karoline Scheffler, Martin D. Shetlar, Bradford W. Gibson

Research output: Contribution to journalArticlepeer-review

137 Scopus citations


Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO2-). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3-nitrotyrosine. In this work, we show that mass spectral patterns associated with 3-nitrotyrosine containing peptides allow identification of peptides containing this modification. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(m-NO2)AR and several peptides containing 3-nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of ions were found for these peptides in addition to the mass shift of +45 Da corresponding to the addition of the nitro group. Specifically, two additional ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that suggest the formation of hydroxylamine and amine derivatives. These latter four components appear to originate by laser-induced photochemical decomposition. MALDI-MS analysis of the synthetic peptide containing 3-nitrotyrosine revealed this same pattern. Post-source decay (PSD) MALDI-time-of-flight (TOF) and collisional activation using a prototype MALDI quadrupole TOF yielded extensive fragmentation that allowed site-specific identification of 3-nitrotyrosine. Conversion of peptides containing 3-nitrotyrosine to 3-aminotyrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abundant sidechain loss under PSD conditions. These observations suggest that MALDI can provide a selective method for the analysis and characterization of 3-nitrotyrosine-containing peptides.

Original languageEnglish (US)
Pages (from-to)439-448
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Issue number4
StatePublished - 2001

Bibliographical note

Funding Information:
This work was supported by a grant funded by University of California and MitoKor (BioSTAR grant #S97-05). We thank Dr. K. Medzihradszky (UCSF) and Dr. A. Verentchikov (Applied Biosystems) for obtaining the MALDI-Q-Tof spectrum on a prototype instrument based on the commercial Mariner platform and the UCSF Mass Spectrometry Facility which is supported in part from the National Center for Research Resources (RR 01614). We also thank Dr. Birgit Schilling for her help in the comparative analysis of intact BSA and nitrated BSA.


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