A rapid and selective mass spectrometric method for the identification of nitrated proteins.
The nitration of protein tyrosine residues represents an important posttranslational modification during development, oxidative stress, and biological aging. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels, from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Identification of the specific targets of protein oxidation was previously accomplished using immunoprecipitation techniques followed by immunochemical detection. Here, we report a totally new approach involving dansyl chloride labeling of the nitration sites which relies on the enormous potential of MS(n) analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and a MS3 analysis. The novel labeling procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.
Amoresano
Angela
A
Department of Clinical and Organic Biochemistry, Federico II University of Naples, Naples, Italy.
Chiappetta
Giovanni
G
Pucci
Piero
P
Marino
Gennaro
G
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D013058:Mass Spectrometry / Q000379:methods*; D008894:Milk Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D009566:Nitrates / Q000032:analysis*; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D008894:Milk Proteins; D009566:Nitrates; D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine; C019253:3-aminotyrosine; C002744:3-nitrotyrosine; D014443:Tyrosine; D014357:Trypsin