Experimental setup.A, topology models of the overexpressed membrane protein GFP fusions. Experimental setup.A, topology models of the overexpressed membrane protein GFP fusions. B, membrane proteins and the GST control were expressed in BL21(DE3)pLysS as GFP fusions from a pET28a+-derived vector as described under “Experimental Procedures.” The A600 of the cultures was measured every hour; pH of the culture was measured every 4 h. Flow cytometry and fluorescence microscopy were used to monitor GFP fusion protein expression and cell morphology. The protein composition of total cell lysates was studied using comparative Western blotting and 2D gel electrophoresis, the protein composition of cytoplasmic aggregates was studied by 1D and 2D gel electrophoresis and in-solution trypsin digest/nano-LC-ESI-MS/MS, and the protein composition of purified cytoplasmic membranes was examined using comparative Western blotting and comparative 2D BN-PAGE. After gel-based proteome analysis, proteins were identified by MALDI-TOF MS/PMF or by peptide sequencing with ESI-Q-TOF nano-LC-ESI-MS/MS. Samuel Wagner et al. Mol Cell Proteomics 2007;6:1527-1550 © 2007 The American Society for Biochemistry and Molecular Biology