Abstract
BackgroundDecoding of the liver proteome is subject of intense research, but hampered by methodological constraints. We recently developed an improved protocol for studying rat liver proteins based on 2-DE-MALDI-TOF-MS peptide mass finger printing.This methodology was now applied to develop a mouse liver protein database.ResultsLiver proteins were extracted by two different lysis buffers in sequence followed by a liquid-phase IEF pre-fractionation and separation of proteins by 2 DE at two different pH ranges, notably 5-8 and 7-10. Based on 9600 in gel digests a total of 643 mouse liver proteins with high sequence coverage (> 20 peptides per protein) could be identified by MALDI-TOF-MS peptide mass finger printing. Notably, 255 proteins are novel and have not been reported so far by conventional two-dimensional electrophoresis proteome mapping. Additionally, the results of the present findings for mouse liver were compared to published data of the rat proteome to compile as many proteins as possible in a rodent liver database.ConclusionBased on 2-DE MALDI-TOF-MS a significantly improved proteome map of mouse liver was obtained. We discuss some prominent members of newly identified proteins for a better understanding of liver biology.
Highlights
Decoding of the liver proteome is subject of intense research, but hampered by methodological constraints
We analyzed mouse liver protein extracts on two narrow pH range immobilized pH gradient (IPG) strips (Figure 2) and processed 24 2-DE gels to yield approximately 9600 gel digests
We investigated the sensitivity, linear range and compatibility of stain for identification by mass spectrometry [8] and used a liquid-phase Isoelectric Focusing (IEF) pre-fractionation in order to reduce the complexity of the samples and to enrich for low-abundance proteins [8]
Summary
Decoding of the liver proteome is subject of intense research, but hampered by methodological constraints. We recently developed an improved protocol for studying rat liver proteins based on 2-DE-MALDI-TOF-MS peptide mass finger printing. This methodology was applied to develop a mouse liver protein database. Life is incompatible without the liver as this organ performs essential metabolic functions. It is of considerable importance that the human and mouse genome display > 99% DNA sequence similarity. This laboratory animal is widely used in biomedical research, for instance, in the evaluation of novel experimental therapeutics.
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