A targeted mass spectrometry-based approach for the identification and characterization of proteins containing α-aminoadipic and γ-glutamic semialdehyde residues.
The site-specific identification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues in proteins is reported. Semialdehydic protein modifications result from the metal-catalyzed oxidation of Lys or Arg and Pro residues, respectively. Most of the analytical methods for the analysis of protein carbonylation measure change to the global level of carbonylation and fail to provide details regarding protein identity, site, and chemical nature of the carbonylation. In this work, we used a targeted approach, which combines chemical labeling, enrichment, and tandem mass spectrometric analysis, for the site-specific identification of AAS and GGS sites in proteins. The approach is applied to in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an untreated biological sample, namely cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two AAA and four GGS residues. Computational evaluation of the identified AAS and GGS sites in GAPDH indicated that these sites are located in flexible regions, show high solvent accessibility values, and are in proximity with possible metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine AAS modification sites which were unambiguously assigned to distinct lysine residues in the following proteins: ATP/ATP translocase isoforms 1 and 2, ubiquinol cytochrome-c reductase core protein 2, and ATP synthase α-subunit.
Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
Juan D
JD
Chavez
William H
WH
Bisson
Claudia S
CS
Maier
D015074:2-Aminoadipic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D005971:Glutamates / Q000737:chemistry* / Q000302:isolation & purification; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry*; D008297:Male; D024101:Mitochondrial Proteins / Q000737:chemistry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D051381:Rats; D011916:Rats, Inbred F344; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D005971:Glutamates; D024101:Mitochondrial Proteins; D015074:2-Aminoadipic Acid; C000061:allysine; C021199:glutamic acid gamma-semialdehyde; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)