Functional proteomics using microchannel plate detectors.
We describe the development of a novel detection system used for the functional imaging of proteins separated on electrophoretic gels. A microchannel plate detector is used here for real-time imaging of low levels of tritiated protein separated by two-dimensional (2-D) electrophoresis. The system employs radioisotope-free, low noise microchannel plates originally developed for photon counting in X-ray astronomy. Using the detector configuration described here, proteins were resolved on mini gels by either one or two-dimensional electrophoresis, transferred onto polyvinylidene difluoride membranes and directly imaged. Tritiated diisopropylfluorophosphate (DFP) was used as a selective label for the serine hydrolase class of enzymes and their distribution in the central nervous system was examined. This survey revealed approximately 24 protein spots by 2-D electrophoresis. We also investigated the relative sensitivity of these proteins towards DFP and found the peptidase, acylpeptide hydrolase to be the most sensitive brain protein towards this reagent. Using a number of different tritiated standards, it was found that the system can image as little as 0.1 Bq/mm(2) of tritium corresponding to 320 attomol of DFP labelled protein/mm(2). Moreover, the system has a wide dynamic range (>10(6)) allowing samples of high and low activity to be quantified on the same gel.
Richards
Paul
P
MRC Toxicology Unit, Leicester, UK. pgr3@le.ac.uk
Lees
John
J
D000818:Animals; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation* / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008297:Male; D020543:Proteome; D051381:Rats; D011916:Rats, Inbred F344; D013223:Statistics as Topic; D014020:Tissue Extracts / Q000737:chemistry
D020543:Proteome; D014020:Tissue Extracts