Proteome Profile of Cytosolic Component of Zebrafish Liver Generated by LC−ESI MS/MS Combined with Trypsin Digestion and Microwave-Assisted Acid Hydrolysis

The zebrafish genome has recently been sequenced and annotated allowing for high-throughput proteomic analysis. Here, we report for the first time a proteomic subset of zebrafish liver, an important organ for metabolizing toxins. Using a newly developed analytical procedure, we have identified 1204 proteins from the cytosolic component of a zebrafish liver tissue sample. Our methods involve cell-compartment fractionation of liver tissue samples, four levels of protein digestion, and off-line two-dimensional liquid chromatography (2-D LC) separations of resultant peptides. Proteins are identified using an electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-QTOF MS/MS), which provides high-resolution and high-accuracy mass measurement of peptide ions and their fragment ions. We demonstrate that greater proteome coverage can be achieved by combining the results obtained from four methods of protein digestion:  three tryptic digests (one in buffer, one in methanol, and another in SDS), and a microwave-assisted acid hydrolysate of the protein extracts. Identified proteinswhich included several groups of established protein biomarkerswere functionally classified. We discuss the functions and implications of these biomarkers within the context of zebrafish toxicology.

Keywords: zebrafish • toxicology • biomarker • liver proteome • LC−ESI MS/MS • microwave-assisted acid hydrolysis