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

Abstract

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.

Analysis of the Zebrafish Proteome during Embryonic Development

“…A more comprehensive shotgun proteomics investigation focused on the identification of protein in livers of adult fish as a resource for toxicological studies (58). Here we provide comprehensive 2D PAGE and shotgun proteomics identifications obtained from whole zebrafish embryos.…”

Strategies for Protein Separation

“…Second, by using coupled SCX-RP columns, separation on both dimensions has to be performed at the same flow rate, which may sacrifice MS detection sensitivity for low-abundance components. As an alternative approach, offline SCX-RP separation has been used to identify more than 1200 proteins from zebrafish liver (Wang et al, 2007).However, the implementation of offline configurations is not always the best option, as extensive offline sample handling increases the overall analysis time and causes sample loss and sensitivity reduction. Dai et al (2005) reported an integrated column composed of SCX and RP where peptides were fractionated by a pH step gradient.…”

Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion

“…Those properties are advantageous for a clean protein identification via MS. Regretfully, its utility as a sample preparation method for protein identification via MS is limited, in that the peptides derived from D site cleavage [17][18][19][20] were not well suited for MS identification considering the sequence length of peptide (average ∼16 AA), in comparison with peptides (∼8 AA) obtained from the conventional urea-assisted protein sample preparation method, which commonly uses trypsin to cleave proteins specifically at the basic residues, arginine and lysine. Given to the drawback above mentioned, commonly a variety of acid cleavage [21,22] was combined with protease digestion for membrane protein analysis. However, such combination method was not investigated thoroughly, aiming at developing a urea free and more efficient protein sample preparation method for MS (MALDI-TOF MS and ESI-MS/MS) identification against the conventional urea-assisted protein sample preparation method for individual protein and the whole proteome analysis.…”