This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Traditional 2-dimentional polyacrylamide gel electrophoresis (2D-PAGE) has a number of well known limitations. It is limited to a fairly narrow range of masses and isoelectric points, it cannot handle highly highly hydrophobic proteins or peptides, and highly basic proteins require special care. Mass estimates are frequently distorted by anomalous migration and/or phosphorylation, deamidation, or glycosylation. We recognized some time ago that some of these problems could be solved by removing the second gel and coupling isoelectric focusing gels to mass spectrometry.[Loo, 1996;Loo, 1997;Loo, 1997;Loo, 2005] We examine complex protein mixtures (whole cell lysates) by MALDI-MS, separated by denaturing isoelectric focusing on immobilized pH gradient gels. Mass analysis is performed directly from dried gels, providing intact mass measurements that relate directly to many 2-D gel analyses; e.g., Western blotting, pulse-chase radiolabeling, etc. In addition, we have recently extended these methods to performing trypsin digestions in-IEF-gel, followed by peptide MS and MS/MS directly from dried gels. This extension adds a third dimension of information to our virtual 2-D gels . Proof-of-principle experiments established that the tryptic peptides generated can be detected by MALDI-MS, and even sequenced by post-source decay. Nevertheless, mass calibration challenges are presented by the uneven gel surface in concert with our commercial time-of-flight instruments ionization source design.
Showing the most recent 10 out of 253 publications
