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

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posted on 05.01.2007, 00:00 by Nan Wang, Lauren MacKenzie, Andrea G. De Souza, Hongying Zhong, Greg Goss, Liang Li
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

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