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.