Chan et al. BMC Genomics 2011, 12:290 http://www.biomedcentral.com/1471-2164/12/290 Page 5 of 11 were put through the PeptideAtlas pipeline to create the final product. The HBPA build resulted in over 1.3 mil- lion peptide-spectrum matches (PSMs) at a FDR of 0.0001. This results in 27,776 distinct peptide sequences at an FDR of 0.0018, mapping to approximately 3000 highly non-redundant proteins at a protein FDR of 0.015. The process of how the peptides are mapped to proteins and the proteins classified is described in detail elsewhere [27]. Further build statistics are listed in Table 2. The sequential addition of the 253 individual samples is depicted in Figure 2. Although each individual experi- ment only contains a few thousand peptides (as depicted by the blue component), the total number of distinct peptides in the entire build continues to increase as more samples are added. The addition of groups of similar samples in succession leads to multiple instances where the total peptide count rises sharply and then flattens out as replicates are added. The PeptideAtlas interface allows the user to explore individual proteins, where they map onto the genome and what MS/MS evidence supports their identity (Fig- ure 3). It also allows one to compare the sequences of honey bee proteins to other bee proteins or even to those in other organisms supported by PeptideAtlas; in particular, peptide evidence for the presence of very clo- sely related isoforms or family members can be dis- played. The usefulness of this feature is exemplified in Figure 4: several similar variants of one protein have been predicted; a large amount of RAW data was searched, but MS/MS data clearly show support for only some of the variants and not others. Honey bees are not a classic model system for mole- cular studies and as such, bee protein functions are relatively poorly annotated. Indeed, each time we have tried to analyze a proteomic dataset from bees Table 2 Summary of the Honey Bee PeptideAtlas 2010-03 build Honey Bee PeptideAtlas 2010-03 Figure 2 Plot showing the cumulative number of distinct peptides added to the Honey Bee PeptideAtlas versus the total number of peptide-spectrum matches (PSMs) about the 0.0001 FDR threshold. Each rectangle represents one of the 253 samples in the atlas. The height of each bar is the cumulative number of distinct peptides a sample is added; the blue component is number of distinct peptides in each sample. The width of the bar denotes the number of PSMs identified above the threshold for each sample. Groups of similar samples cause the cumulative number of distinct peptides to level off, but as a new sample type is added, the number of additional distinct peptides added to the build increases significantly. [5,11,13], we have been forced to re-generate Gene Ontology (GO) [28] classifications for the proteins of interest. GO is a controlled vocabulary describing the molecular function, biological process and cellular component for gene products, where the same terms are used across all species. In order to provide an ontological classification scheme for bee proteins as an additional resource to the community, BLAST2GO [25] was used to assign tentative annotations to honey bee proteins based on the closest sequence homolog for which GO assignments are available. A total of 9009 sequences can be matched to at least one GO term, or about 37% of the sequences in the Honey Bee PeptideAtlas. The majority remains unmatched, pri- marily because the input sequences include ones from earlier annotations of the genome which have been eliminated in later versions; in searching our MS data against them we saw that some were falsely excluded since they match peptide spectra, however, we inevita- bly include protein sequences that are not actually translated. Another reason would be the lack of well- annotated insect proteins (e.g., relative to human and mouse) for bee proteins to match against. In perform- ing manual checks, even highly abundant and impor- tant bee sequences such as major royal jelly proteins (the larval food source) and odorant binding proteins (soluble transporters of hydrophobic odor molecules) are not matched to any GO terms. Build Total Experiments Total ms runs Spectra searched PSMs above threshold Distinct peptides Distinct proteins Mayu Analysis PSM Peptide Protein 253 1,594 5,601,751 1,339,806 27,776 3,009 TP_PSM: 1,339,586 FP_PSM 110 FDR: 0.0000821 TP_pepID: 27,725, FP_pepID 51 FDR 0.00180 TP_protID: 3009, FP_protID 50 FDR: 0.0154