Chan et al. BMC Genomics 2011, 12:290 http://www.biomedcentral.com/1471-2164/12/290 Page 4 of 11 the decoy identifications. These filtered results were loaded into the PeptideAtlas database as build “Honeybee 2010-03” and can be downloaded and browsed in the usual manner [10]. Predicting protein function with BLAST2GO Since functional annotations are very incomplete for honey bee, we have attempted to greatly expand the protein annotations using the Gene Ontology (GO). The GO system organizes all protein annotations into a hier- archical structure of increasing granularity, with three separated root categories: molecular function, biological process, and cellular component. All proteins were pro- cessing through the BLAST2GO program [25] which yields a set of GO annotations for each protein based on homology to proteins from other species as deter- mined by BLAST [26]. We used this software according to the default protocols and settings: BLAST searches were conducted for each protein (BLASTp, nr database, HSP cutoff length 33, report 20 hits, maximum e-Value 1e-10), followed by mapping and annotation (e-Value hit filter 1e-10, annotation cutoff 55, GO weight 5, HSP- hit coverage cutoff 20). The results of the BLAST2GO mapping may be downloaded at the same URL as pre- viously provided for other data products described above. Results The Honey Bee PeptideAtlas (HBPA) was assembled from MS/MS collected over four years from all three castes, larvae and virtually all adult honey bee tissues as described in Methods and as outlined in Figure 1. The data were searched in a first-pass genomic search to identify a set of putative new protein sequences. All honey bee proteins were annotated via BLAST2GO. All MS/MS data along with the new annotation information Figure 1 Creating the Honey Bee PeptideAtlas. Honey bee tissues of different ages, sexes, and disease states were analyzed by MS using the high accuracy LTQ-OrbitrapXL or LTQ-FT. To improve proteome depth, we used samples which were biological replicates (i.e. same experiment on more than one bee), technical replicates (i.e. one bee sample analyzed more than once by MS), and employed various fractionation techniques. The RAW files were put through the PeptideAtlas pipeline. The database used for searching the MS data contained sequences from various publicly available sources, as well as novel and corrected proteins derived from the proteogenomics effort reported in this article. Using BLAST2GO, GO terms were annotated to proteins where appropriate. This result of this work comprises the Honey Bee PeptideAtlas.