title: Identification of carbonylated peptides by tandem mass spectrometry using a precursor ion-like scan in negative ion mode.
abstract: Reactive oxygen species (ROS) can oxidize proteins at almost any amino acid residue. Whereas some modifications are reversible within the cells, the higher oxidation states are especially irreversible. These irreversible post translational modifications are widely used as biomarkers of oxidative stress, such as protein carbonylation, which refers to aldehydes, ketones and lactams as 'reactive carbonyl groups'. This study relied on a set of synthetic peptides containing a C-terminal aldehyde (arginal) or modification with pyruvic acid (ketone) or 4-hydroxynonenal (aldehyde) at lysine or histidine residues, as well as peptides containing pyroglutamic acid (oxidation product of proline) and 2-amino-3-butyric acid (oxidation product of threonine). The carbonylation sites were specifically derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the fragmentation behavior of the products investigated in electrospray ionization (ESI-) MS. Importantly, the DNPH-labeled carbonylated peptides showed favorable ionization behaviors in negative ion mode ESI, providing a sensitive detection method. Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, pulsed Q dissociation provided three diagnostic ions at m/z values 152.0, 163.1 and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect the carbonylated model peptides in a spiked tryptic digest of bovine serum albumin and a complex protein mixture obtained from HeLa cells.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany. ravi.bollineni@bbz.uni-leipzig.de
authors.forename: Ravi Ch
authors.initials: RCh
authors.lastname: Bollineni
authors.forename: Maria
authors.initials: M
authors.lastname: Fedorova
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006367:HeLa Cells; D006801:Humans; D007477:Ions / Q000032:analysis / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010659:Phenylhydrazines / Q000737:chemistry / Q000494:pharmacology; D050050:Protein Carbonylation; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007477:Ions; D010455:Peptides; D010659:Phenylhydrazines; C004787:2,4-dinitrophenylhydrazine; D012710:Serum Albumin, Bovine