title: Evaluation of a method for nitrotyrosine site identification and relative quantitation using a stable isotope-labeled nitrated spike-in standard and high resolution fourier transform MS and MS/MS analysis.
abstract: The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO-), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z=181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA. kseeley@usf.edu.
authors.forename: Kent W
authors.initials: KW
authors.lastname: Seeley
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA. afertig@mail.usf.edu.
authors.forename: Alison R
authors.initials: AR
authors.lastname: Fertig
authors.affiliation: Training Institute, Thermo Fisher Scientific, 1400 Northpoint Parkway, Ste 10., West Palm Beach, FL 33407, USA. craig.dufresne@thermofisher.com.
authors.forename: Craig P
authors.initials: CP
authors.lastname: Dufresne
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA. costapinho@mail.usf.edu.
authors.forename: Joao P C
authors.initials: JP
authors.lastname: Pinho
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA. smstevens@usf.edu.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Stevens
mesh_terms: D000803:Angiotensin I / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005583:Fourier Analysis; D007553:Isotope Labeling; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000737:chemistry; D051381:Rats; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D009587:Nitrogen Isotopes; D030421:Peroxynitrous Acid; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine; D000803:Angiotensin I
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