title: N-Terminal Peptide Detection with Optimized Peptide-Spectrum Matching and Streamlined Sequence Libraries.
abstract: We identified tryptic peptides in yeast cell lysates that map to translation initiation sites downstream of the annotated start sites using the peptide-spectrum matching algorithms OMSSA and Mascot. To increase the accuracy of peptide-spectrum matching, both algorithms were run using several standardized parameter sets, and Mascot was run utilizing a, b, and y ions from collision-induced dissociation. A large fraction (22%) of the detected N-terminal peptides mapped to translation initiation downstream of the annotated initiation sites. Expression of several truncated proteins from downstream initiation in the same reading frame as the full-length protein (frame 1) was verified by western analysis. To facilitate analysis of the larger proteome of Drosophila, we created a streamlined sequence library from which all duplicated trypsin fragments had been removed. OMSSA assessment using this "stripped" library revealed 171 peptides that map to downstream translation initiation sites, 76% of which are in the same reading frame as the full-length annotated proteins, although some are in different reading frames creating new protein sequences not in the annotated proteome. Sequences surrounding implicated downstream AUG start codons are associated with nucleotide preferences with a pronounced three-base periodicity N1^G2^A3.
authors: Department of Biology, Wesleyan University , Middletown, Connecticut 06459, United States.|Department of Mathematics and Computer Science, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Brynne E
authors: BE
authors: Lycette
authors: Department of Biology, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Jacob W
authors: JW
authors: Glickman
authors: Department of Biology, Wesleyan University , Middletown, Connecticut 06459, United States.|Department of Mathematics and Computer Science, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Samuel J
authors: SJ
authors: Roth
authors: Department of Biology, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Abigail E
authors: AE
authors: Cram
authors: Department of Biology, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Tae Hee
authors: TH
authors: Kim
authors: Department of Mathematics and Computer Science, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Danny
authors: D
authors: Krizanc
authors: Department of Biology, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Michael P
authors: MP
authors: Weir
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D018387:Codon, Initiator; D030562:Databases, Protein / Q000592:standards*; D029721:Drosophila Proteins / Q000032:analysis*; D005656:Fungal Proteins / Q000032:analysis*; D058977:Molecular Sequence Annotation; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods* / Q000592:standards; D016364:Reading Frames; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D018387:Codon, Initiator; D029721:Drosophila Proteins; D005656:Fungal Proteins; D010455:Peptides
keywords: Mascot; OMSSA; peptide mass spectrometry; streamlined sequence libraries