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 proteinswhich included several groups of established protein biomarkerswere 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