N-Terminal Peptide Detection with Optimized Peptide-Spectrum Matching and Streamlined Sequence Libraries.
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.
['Lycette|Brynne E|BE|', 'Glickman|Jacob W|JW|', 'Roth|Samuel J|SJ|', 'Cram|Abigail E|AE|', 'Kim|Tae Hee|TH|', 'Krizanc|Danny|D|', 'Weir|Michael P|MP|']
D000465:Algorithms D000595:Amino Acid Sequence D000818:Animals D018387:Codon, Initiator D030562:Databases, Protein D029721:Drosophila Proteins D005656:Fungal Proteins D058977:Molecular Sequence Annotation D010455:Peptides D040901:Proteomics D016364:Reading Frames D012015:Reference Standards D053719:Tandem Mass Spectrometry
streamlined sequence libraries OMSSA Mascot peptide mass spectrometry