Sub-proteome differential display: single gel comparison by 2D electrophoresis and mass spectrometry.
Two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) have been used in comparative proteomics but inherent problems of the 2D electrophoresis technique lead to difficulties when comparing two samples. We describe a method (sub-proteome differential display) for comparing the proteins from two sources simultaneously. Proteins from one source are mixed with radiolabelled proteins from a second source in a ratio of 100:1. These combined proteomes are fractionated simultaneously using column chromatographic methods, followed by analysis of the pre-fractionated proteomes (designated sub-proteomes) using 2D gel electrophoresis. Silver staining and (35)S autoradiography of a single gel allows precise discrimination between members of each sub-proteome, using commonly available computer software. This is followed by MS identification of individual proteins. We have demonstrated the utility of the technology by identifying the product of a transfected gene and several proteins expressed differentially between two renal carcinoma proteomes. The procedure has the capacity to enrich proteins prior to 2D electrophoresis and provides a simple, inexpensive approach to compare proteomes. The single gel approach eliminates differences that might arise if separate proteome fractionations or 2D gels are employed.
Spandidos
Athanasia
A
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England, UK.
Rabbitts
Terence H
TH
D000818:Animals; D001345:Autoradiography; D002292:Carcinoma, Renal Cell / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007525:Isoelectric Focusing; D007680:Kidney Neoplasms / Q000378:metabolism; D008164:Luminescent Proteins; D013058:Mass Spectrometry / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D008164:Luminescent Proteins; D020543:Proteome; D049452:Green Fluorescent Proteins