Method development for protein enrichment in gel-free proteomic analysis of whole fish

Organ-specific proteomics of larval fish is limited by difficulties in dissection. Thus, method development for efficient proteomic analysis of whole fish is required to investigate changes in larvae physiology. Proteome assessment of whole fish is hampered by the presence of high-abundant proteins, which interfere with the detection of low-abundant proteomic components, and consequently hide physiologically significant changes restricted to a certain cell type. Several strategies exist to reduce sample complexity and extend the range of proteins detected. For example, in 2-D gel based proteomics, narrower pH–range IPG strips and larger gels can increase the detection of lower-abundance proteins. Our aim is to develop a robust protein extraction method from whole fish larvae to study changes in protein expression using label-free shotgun proteomics. Different protein extraction methods were compared for their ability to enhance protein enrichment in samples of Atlantic salmon larvae (~200 mg). Manual sample disruption was compared with mechanical homogenization, and direct protein extraction (Urea/Thiourea/Tris) was compared with solubility-based protein fractionation (NaCl/Tris + Urea/Thiourea/Tris). SDS-PAGE analysis of sequential fractionation revealed distinct protein profiles. Preliminary studies on whole Atlantic salmon larvae using manual disruption and direct protein extraction identified 432 proteins. Ongoing shotgun proteomic analysis will determine the effectiveness of this approach to increase proteome coverage.