Analysis of the cattle liver proteome by high-sensitive liquid chromatography coupled with mass spectrometry method.
The present chapter describes methods for the separation and identification of proteins in liver metabolism through a comparison of the protein expression profiles of the two breeds taken into account as a model: Holstein Friesian and Chianina cattle. The liver has received special attention, containing as it does, enzymes involved in energy generation, carbohydrate, lipid, amino acid, and xenobiotic metabolism, as well as proteins involved in polypeptide synthesis, folding, and cell structure. The first step in the procedure is the preparation of purified protein fractions from liver tissues, followed by sample preparation for 2-DE analysis in order to identify proteins which could be differentially expressed in the livers of the two breeds and relate them to different liver functions. Data can be then statistically elaborated with cluster analysis, which stressed the up-/on-regulation trend of these proteins. Quantitative data can be used to perform a two-way hierarchical cluster analysis of the 39 differentially expressed protein spots, either up- or on-regulated in Chianina versus Holstein Friesian liver samples. Thus, spots from 2-DE maps can be carefully excised from the gel and subjected to in-gel trypsin digestion and analyzed by tandem mass spectrometry in their contents.
Department of Environmental Sciences, University of Tuscia, Largo dell'Università snc, Viterbo, Italy. timperio@unitus.it
Anna Maria
AM
Timperio
Gian Maria
GM
D'Amici
Lello
L
Zolla
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D015870:Gene Expression; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000737:chemistry
D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin