Distribution of cuticular proteins in different structures of adult Anopheles gambiae

• Tandem mass spectrometry was used to examine proteins in Johnston's organs, antennae, eye lenses, legs, and wings from adult Anopheles gambiae.
• Particular attention was paid to cuticular proteins (CPs) where we had matches to 142 previously annotated CPs.
• These data were compared to four published studies to learn whether any structures had unique proteins.
• In addition, we identified 467 non-cuticular proteins.
• A quantitative assessment of relative abundance was carried out using normalized spectral counts.

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins.

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