This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objective of this project is to establish a set of protease digest conditions suitable for proteomic and mass spectrometry (MS) based analysis of membrane proteins. The motivation for pursuing this work is not only the tremendous importance of membrane proteins, but also the growing application of proteomics methods for the identification, quantitation, and characterization of proteins in general. To achieve the objective we are pursuing two aims. Aim 1 tests protease digest conditions using two types of model membrane proteins (bacteriorhodopsin and phospholipase A2) with the goal of identifying an optimized set of conditions by varying methanol solvent concentration, temperature, enzyme (chymotrypsin, trypsin, or both), detergent additives, and time. The determination of an improved protocol will be based on a Mascot score (spectral assignment) and on sequence coverage. In Aim 2 the two resulting optimized protocols will be tested against proteins from collaborators who are active COBRE faculty working on membrane proteins. We have already been working with these collaborators on membrane protein studies and will apply the new methods to their samples. These samples include work on membrane associated proteins, cell wall proteins, plasmodesmata associated proteins, and others. If Aims 1 and 2 are completed in a timely manner, then we will pursue an extension to the project that involves the proteomic analysis, using the newly developed methods, of membrane proteins from E. coli and from CHO cells.