Bidimensional tandem mass spectrometry for selective identification of nitration sites in proteins.
Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. 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. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn 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 an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.
Amoresano
Angela
A
Department of Organic Chemistry and Biochemistry and School of Biotechnological Sciences, Federico II University of Naples, 80126 Napoli, Italy.
Chiappetta
Giovanni
G
Pucci
Piero
P
D'Ischia
Marco
M
Marino
Gennaro
G
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003619:Dansyl Compounds / Q000737:chemistry; D008892:Milk / Q000737:chemistry; D008894:Milk Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
D003619:Dansyl Compounds; D008894:Milk Proteins; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine; D014357:Trypsin; C029038:dansyl chloride