title: Pitfalls in protein quantitation using acid-catalyzed O18 labeling: hydrolysis-driven deamidation.
abstract: Proteolysis combined with O(18) labeling emerged recently as a powerful tool for quantitation of proteins for which suitable internal standards cannot be produced using molecular biology methods. Several recent reports suggested that acid-catalyzed O(18) labeling may be superior to the commonly accepted enzymatic protocol, as it may allow more significant spacing between the isotopic clusters of labeled and unlabeled peptides, thereby eliminating signal interference and enhancing the quality of quantitation. However, careful examination of this procedure reveals that the results of protein quantitation assisted by acid-catalyzed O(18) labeling are highly peptide-dependent. The inconsistency was found to be caused by deamidation of Asn, Gln, and carbamidomethylated Cys residues during prolonged exposure of the proteolytic fragments to the acidic environment of the labeling reaction, which translates into a loss in signal for these peptides. Taking deamidation into account leads to a significant improvement in the consistency of quantitation across a range of different proteolytic fragments.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts 01003, United States.
authors.forename: Shunhai
authors.initials: S
authors.lastname: Wang
authors.forename: Cedric E
authors.initials: CE
authors.lastname: Bobst
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000143:Acids / Q000737:chemistry*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003641:Deamination; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D010103:Oxygen Isotopes / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D014168:Transferrin / Q000032:analysis / Q000378:metabolism
chemical_list: D000143:Acids; D010103:Oxygen Isotopes; D011506:Proteins; D014168:Transferrin; D005973:Glutamine; D001216:Asparagine