Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

Abstract

The exoskeleton of arthropods, called cuticle, is a biological composite material consisting principally of N–acetylglucosamine polymer embedded in cuticular proteins (CPs). The CPs have been studied and characterized by mass spectrometry in several cuticular structures and in many arthropods. Such analyses were usually carried out by protein extraction using SDS detergent followed by electrophoresis, allowing detection and identification of a large number of CPs. To build a repertoire of cuticular structures from Bombyx mori, Apis mellifera and Anopheles gambiae we avoided the use of SDS and electrophoresis. Based on the combination of hexafluoroisopropanol solvent and of a surfactant solution fully compatible with MS, we successfully identified a high number of CPs in wings, legs and antennae of An. gambiae, as well as in the thoracic integument cuticle of Ap. mellifera pupae. The complete exoskeleton analysis of B. mori larvae allowed us to identify 85 CPs from the cuticle of a single larva. Finally, we tested our novel proteomics approach on cuticles left behind after the molt from the fourth instar of the pea aphid Acyrthosiphon pisum. Analysis of these cast cuticles allowed us to identify 100 Ac. pisum CPs as authentic constituents of the cuticle. These correspond to 68% of the total putative CPs previously annotated for this pea aphid. While this paper analyzes only the recovered cuticular proteins, peptides from many other proteins were also detected.

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