Sequential protein extraction as an efficient method for improved proteome coverage in larvae of Atlantic salmon ( Salmo salar )

Abstract

Understanding diet- and environmentally induced physiological changes in fish larvae is a major goal for the aquaculture industry. Proteomic analysis of whole fish larvae comprising multiple tissues offers considerable potential but is challenging due to the very large dynamic range of protein abundance. To extend the coverage of the larval phase of the Atlantic salmon (Salmo salar) proteome, we applied a two-step sequential extraction (SE) method, based on differential protein solubility, using a nondenaturing buffer containing 150 mM NaCl followed by a denaturing buffer containing 7 M urea and 2 M thiourea. Extracts prepared using SE and one-step direct extraction were characterized via label-free shotgun proteomics using nanoLC-MS/MS (LTQ-Orbitrap). SE partitioned the proteins into two fractions of approximately equal amounts, but with very distinct protein composition, leading to identification of ∼40% more proteins than direct extraction. This fractionation strategy enabled the most detailed characterization of the salmon larval proteome to date and provides a platform for greater understanding of physiological changes in whole fish larvae. The MS data are available via the ProteomeXchange Consortium PRIDE partner repository, dataset PXD003366.

Chronic β2‐adrenoceptor agonist treatment alters muscle proteome and functional adaptations induced by high intensity training in young men

“…; Nuez‐Ortin et al . , ). Proteins were extracted in progressively denaturing buffers (150 m m NaCl, 50 m m Tris, pH 8.0 followed by 7 m urea, 2 m thiourea, 50 m m Tris, pH 8.0).…”

“…), whole fish larvae (Nuez‐Ortin et al . ) and in white muscle (Nuez‐Ortin et al . ) and skeletal muscle (Barbé et al .…”

Comprehensive nuclear proteome of Arabidopsis obtained by sequential extraction

“…In this study, to improve the quality and quantity of the plant nuclear proteome, we used a buffer with a high concentration of sucrose to purify nuclei and then conducted solubility-based fractionation to increase proteome coverage [43,44]. We identified 1539 different proteins from the nuclei of Arabidopsis cultured cells and two novel NE proteins.…”