Toward an Upgraded Honey Bee (Apis mellifera L.) Genome Annotation Using Proteogenomics

Abstract

The honey bee is a key pollinator in agricultural operations as well as a model organism for studying the genetics and evolution of social behavior. The Apis mellifera genome has been sequenced and annotated twice over, enabling proteomics and functional genomics methods for probing relevant aspects of their biology. One troubling trend that emerged from proteomic analyses is that honey bee peptide samples consistently result in lower peptide identification rates compared with other organisms. This suggests that the genome annotation can be improved, or atypical biological processes are interfering with the mass spectrometry workflow. First, we tested whether high levels of polymorphisms could explain some of the missed identifications by searching spectra against the reference proteome (OGSv3.2) versus a customized proteome of a single honey bee, but our results indicate that this contribution was minor. Likewise, error-tolerant peptide searches lead us to eliminate unexpected post-translational modifications as a major factor in missed identifications. We then used a proteogenomic approach with ~1500 raw files to search for missing genes and new exons, to revive discarded annotations and to identify over 2000 new coding regions. These results will contribute to a more comprehensive genome annotation and facilitate continued research on this important insect.

Odorant cues linked to social immunity induce lateralized antenna stimulation in honey bees (Apis mellifera L.)

“…Interestingly, 230 of the identified proteins are ones that were discarded from the first Official Gene Set (OGSv1.0), apparently in error. We described this phenomenon previously31 and this finding offers secondary confirmation. A further 15 proteins are new sequences which we identified in the same previous proteogenomic effort.…”

“…Dried protein pellets were resuspended in 50 mM ammonium bicarbonate buffer (1% sodium deoxycholate) and protein concentration was determined using a bicinchoninic assay (Pierce). Protein was reduced, alkylated, digested and analyzed on an LC-ESI-MSMS system (Easy nLC-1000 coupled to a Bruker Impact II mass spectrometer) as described in our previous publication31, except we loaded 2.5 μg (based on protein quantitation), the LC gradient was 165 min and MS/MS frequency was set to 18 Hz (see embedded microTOFQImpactAcquisition.method files within PXD005242 for further details).…”

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…Cell lysates were prepared and fractionated, SILAC/MS analyses were performed, and 17S rRNA was quantified using established methods (10,14,50,51), as detailed in SI Methods.…”

Results of follow-up experiments to “Odorant cues linked to social immunity induce lateralized antennal stimulation in honey bees (Apis mellifera L.)”

“…To improve proteome coverage in the current study, we fractionated the peptides from left and right antennae from 4 highly hygienic colonies and repeated the comparison (Figure 2). This time, we identified 3,114 unique proteins (a 69% improvement), which is among the highest proteome coverage achieved in honey bees to date (7). However, we still did not identify significant differences.…”