Characterization of Zebrafish Cardiac Proteome Using Online pH Gradient SCXRP HPLCMS/MS Platform

Two-dimensional HPLC coupled with tandem MS (MS/MS) has become a mainstream technique in the shotgun proteomics for large-scale identification of proteins from biological samples. This powerful technology provides speed, sensitivity, and dynamic range which are essential to probe complex peptide mixtures from proteomic samples. Herein we present a pH gradient SCX–RP 2D HPLC–MS/MS method designed to improve the peptide resolution and protein identification from complex proteomic samples. The comparison between the pH gradient SCX–RP 2D HPLC method and traditional salt gradient SCX–RP method was presented. A two-step sample prefractionation method utilizing microwave-assisted tryptic digestion to improve the identification of insoluble proteins was also introduced. This novel 2D HPLC–MS/MS method was applied to the heart proteomic sample of the zebrafish, Danio rerio , to provide comprehensive cardiac proteomic profiling of this important model organism for cardiovascular and environmental toxicology studies.