For complex protein mixtures, including cell lysates and serum, containing hundreds and even thousands of proteins, 2-D gel-electrophoresis separation (2D-PAGE) is the standard method used to separate and isolate individual proteins for analysis. In addition to being useful for identifying individual proteins, it is also used to monitor changes in the amount of a specific protein or proteins as an indication of the onset of a disease in tissue or to study how disease develops and how pathology is affected by its environment and by methods to treat it. We will develop a capillary equivalent to the standard 2D-PAGE method that offers several improvements compared to 2D-PAGE, including automation, quantitative measurement of individual proteins, dramatically decreased run time, reproducible size and pI measurement for protein identification, and an interface to deposit specific proteins of interest onto a MALDI plate for TOP mass spectrometry identification. Innovations include a scheme to couple isoelectric focusing (CIEF) in a glass micro-fluidic chip channel with gel electrophoresis (CGE) in a capillary array with fluorescence detection for automated two-dimensional electrophoresis analogous to 2D-PAGE and a scheme to couple the output of a capillary array to a MALDI plate for """"""""off-line"""""""" protein identification. In Phase I, both coupling schemes will be developed and tested. In addition, pre-fractionation of samples to improve sensitivity and separation efficiency will be investigated. This will be a significant advance for the analysis of whole cell samples, serum, and other bodily fluids for clinical diagnosis and research studies of cancerous and pre-cancerous tissue. A MALDI interface to a capillary array will be the first demonstration of this capability. ? ?
| Bernard, Richard; Loge, Gary (2009) Poly(2-ethyl-2-oxazoline) as a sieving matrix for SDS-CE. Electrophoresis 30:4059-62 |
