title: N-Terminal amino acid side-chain cleavage of chemically modified peptides in the gas phase: a mass spectrometry technique for N-terminus identification.
abstract: Although genome databases have become the key for proteomic analyses, de novo sequencing remains essential for the study of organisms whose genomes have not been completed. In addition, post-translational modifications present a challenge in database searching. Recognition of the b or y-ion series in a peptide MS/MS spectrum as well as identification of the b1 - and yn-1 -ions can facilitate de novo analyses. Therefore, it is valuable to identify either amino-acid terminus. In previous work, we have demonstrated that peptides modified at the epsilon-amino group of lysine as a t-butyl peroxycarbamate derivative undergo free radical promoted peptide backbone fragmentation under low-energy collision-induced dissociation (CID) conditions. Here we explore the chemistry of the N-terminal amino group modified as a t-butyl peroxycarbamate. The conversion of N-terminal amines to peroxycarbamates of simple amino acids and peptides was studied with aryl t-butyl peroxycarbonates. ESI-MS/MS analysis of the peroxycarbamate adducts gave evidence of a product ion corresponding to the neutral loss of the N-terminal side chain (R), thus identifying this residue. Further fragmentation (MS3) of product ions formed by N-terminal residue side-chain loss (-R) exhibited an m/z shift of the b-ions equal to the neutral loss of R, therefore labeling the b-ion series. The study was extended to the analysis of a protein tryptic digest where the SALSA algorithm was used to identify spectra containing these neutral losses. The method for N-terminus identification presented here has the potential for improvement of de novo analyses as well as in constraining peptide mass mapping database searches.
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Almary
authors.initials: A
authors.lastname: Chacon
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Masterson
authors.forename: Huiyong
authors.initials: H
authors.lastname: Yin
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
authors.forename: Ned A
authors.initials: NA
authors.lastname: Porter
mesh_terms: D000588:Amines / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D002219:Carbamates / Q000138:chemical synthesis / Q000737:chemistry; D005740:Gases; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D010545:Peroxides / Q000138:chemical synthesis / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
chemical_list: D000588:Amines; D000596:Amino Acids; D002219:Carbamates; D005740:Gases; D010455:Peptides; D010545:Peroxides