Chan et al. BMC Genomics 2011, 12:290 http://www.biomedcentral.com/1471-2164/12/290 Page 7 of 11 Figure 5 Comparing the honey bee and fruit fly. (a) Chart showing the relative occurrences of GO terms for honey bee and fruit fly. Each bar represents the log ratio of the number of proteins annotated with a given category for fly vs. bee. Black bars represent observed proteins in PeptideAtlas; Grey bars represent all annotated proteins. (b) Pie charts representing protein (I, II) and gene (III, IV) coverage in the Honey Bee (I, III) and Fly (II, IV) PeptideAtlases. common of which is thought to be the omission of some post-translational modifications in the database search parameters. However, incomplete gene annota- tion is another likely cause of unassigned spectra: if the gene has not been identified or has been mis-annotated then the relevant peptide that could match to the spec- tra in question might not be present in the database. Even Saccharomyces cerevisiae genes are not completely annotated but few unannotated proteins can be found, even with deep proteome coverage [29]; for organisms with relatively short histories in genomic research, such as honey bees, the gene annotation is still quite fluid [30]. Furthermore, sometimes predicted genes or experi- mentally observed ones (e.g., by expressed sequence tags) are not translated in silico and therefore not placed into publically accessible protein databases. Given that MS data is not searched by BLAST against a nucleic acid database but by protein database only, this likely represents a significant source of “missing” proteins - an oversight that we hope to address with this proteoge- nomics effort. By searching a six-frame translation of the entire bee genome we have previously been able to identify several apparently real proteins expressed from unannotated genes (i.e., ones which are not in protein databases) [12] so here we undertook a more systematic and larger-scale re-annotation of bee proteins using MS/ MS data (Figure 6). Spectra matching peptides in the existing protein database (27,256 unique sequences, we call these “pre-existing peptides”) were separated from the unmatched spectra, using a Perl script. The latter was searched against the ORFs of a six-frame translation of the honey bee genome, resulting in 13,240 peptides (we call these “new peptides”) matching to 14,878 ORFs. All spectra were searched against these ORFs, and after omitting the single hits, 186 ORFs remain (Additional File 1) that can be divided into two groups: “Group A” being matched by both pre-existing and new peptides, i. e. corrections of previously annotated proteins, and “Group B” being matched only be new peptides, i.e. novel proteins. Note that Group A proteins likely include mis-annotations of intron-exon boundaries or, possibly, an exon that was entirely missed previously. Figure 6 Annotation and reannotation of bee genes. MS2 data were searched against a 6-frame translation of all ORFs from the honey bee genome, ignoring matches to contaminants, partial/non- tryptic, short (≤ 5 residues), and end-of-sequence peptides. Peptides that match already annotated proteins are in ➀, and those that did not are in ➁. In ➂, the ORFs matched by ➁ were compiled. In ➃, we tested which peptides can be matched to the ORFs in ➂. The ORFs which are matched only by peptides of ➁ are novel proteins (➄). The ORFs which are matched by peptides from both ➀ and ➁ are corrections of already annotated proteins (➅); most commonly, these were residues that had previously been falsely classified as introns, where current MS spectra now confirm their expression. “GROUP A” and “GROUP B” proteins refer to corrected and new proteins, respectively (see manuscript text).