Chan et al. BMC Genomics 2011, 12:290 http://www.biomedcentral.com/1471-2164/12/290 Page 6 of 11 Figure 3 Screenshot of a protein view within PeptideAtlas for protein GB12497-PA. The general protein view has several collapsible sections that provide information about the protein. Section I provides known aliases and descriptions of the protein including functional annotations from our BLAST2GO results, while Section II depicts the distribution of observed and unlikely peptides in a graphical format. Section III shows the full amino acid sequence with observed parts colored in red. Section IV lists individual observed peptides and their attributes. These assignments are integrated into the PeptideAtlas for each protein and hyperlinked to further information regarding the particular term. The annotations appear in the protein summary page for each protein, and one may search for all proteins associated with a given term via the main PeptideAtlas search page. For example, a search for “photoreceptor” yields 23 distinct proteins, which contained the query term either in the protein description or GO annotation. Each description is linked to a page pertaining to the protein of interest with further information regarding its MS evidence, fre- quency of observation, proteotypic peptides, and more. Based on these assignments, we then compared the most-commonly matched GO terms between the bee and fruit fly - their whole proteomes and just the non- redundant sequences represented in the respective Pep- tideAtlases (Figure 5a). Interestingly, a few striking observations emerge from this comparison: e.g., flies seem to have a much larger repertoire of proteins involved in redox metabolism, perhaps reflecting their need to survive in a wider variety of environments and on more diverse food sources than honey bees. On the other hand, ribosomal and other ribonucleoproteins seem to be proportionally more abundant in bees. How- ever this enrichment likely reflects the much greater representation of individual tissues in the bee PeptideA- tlas, which contains MS data from systematic dissections of the various body parts. This would be virtually impossible in the much smaller fruit fly. A comparison of overall gene and protein coverage in the Fly and Honey Bee PeptideAtlases is also illuminat- ing and likewise suggests that greater coverage of var- ious tissues, life stages and castes in honey bee has enabled wider coverage of closely related proteins. Cov- erage of ‘possibly distinguished’ and ‘subsumed’ genes (Figures 5bi and 5bii) proteins (Figure 5biii and 5biv) relative to canonical proteins is higher in bees, as one might expect from greater diversity of samples analyzed. The overall fraction of bee proteins represented in Pep- tideAtlas is significantly less than for fly partly because there is simply more data available for Drosophila but also because we have chosen to use a much larger, more inclusive protein library for Honey Bee than the NCBI library, which contains 9,759 proteins (at the time of writing). While a significant fraction of the 24,558 pro- teins in the library used in PeptideAtlas are likely not real, we have taken this approach since MS data can provide evidence for proteins that would otherwise be excluded. Correction and identification of new proteins Typically only 10 to 30% of the fragment spectra from a shotgun proteomics experiment are matched to a pep- tide when searched against a database of relevant taxo- nomic constraint. Many apparently high quality spectra are not matched for a number of reasons, the most Figure 4 Screenshot of a PeptideAtlas sequence alignment of three similar proteins. The sequences are aligned with ClustalW, whose consensus string is show below the sequences; an asterisk indicates identity for all proteins. Sequence is colored blue or green where observed peptides are seen. There is no independent evidence that the top protein is detected, while there is significant evidence that the bottom form is detected.