title: Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification.
abstract: Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.
authors.affiliation: CST-9, BN-2, MS M888, Chemical Science & Technology Division, Los Alamos National Laboratory, New Mexico, USA. xchen@telomere.lanl.gov
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: L M
authors.initials: LM
authors.lastname: Smith
authors.forename: E M
authors.initials: EM
authors.lastname: Bradbury
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D007554:Isotopes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D007554:Isotopes