Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the Dl and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.

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Presynaptic calcium channels are key regulators of neurotransmitter release. Oocyte expression studies suggest that cysteine string proteins are essential subunits or modulators of these channels. Subcellular fractionation revealed that cysteine string proteins copurify with synaptic vesicles. An average vesicle had eight protein monomers with both the amino and carboxyl termini detected on the cytoplasmic face. Thus, docked synaptic vesicles may regulate presynaptic calcium channels and neurotransmitter release.

Classical neurotransmitters such as acetyl- conting at 3 pM degenerate primers (5'-GCXYTXYTX-YTXGAXAAXATGYT-3' and 5'-ZTCXGCPATXGCZT-AXAC-3', where X = A, C, G, and T; Y = C and T; Z = A and G; and P = A, G, and T) and Taq polymerase under the following conditions: 4 min at 94°C and then 35 cycles of 1 min at-94C, 2 min at 55TC, and 3 min at 72TC. The products were separated by electrophoresis, and a 1.1-kb fragment was subcloned and sequenced on both strands by the method of chain termination (9). For PCR amplification of rat sequences, 1 pig of poly(A)+ rat spinal cord RNA was reversetranscribed and amplified as above by using the degenerate primers 5'-GCXTTYYTXGAZCCXACXAT-3' and 5'-AY-XAZXGCPATXCCZAAZCA-3'. This reaction produced a 260-bp fragment that was then excised from a low-melting temperature agarose gel, subcloned, and sequenced.Northorn Blot Analysis. Poly(A)+ RNA was separated by electrophoresis through formaldehyde-agarose; blotted to nitrocellulose; hybridized overnight at 420C in 50%9 formamide containing 5x SSPE (0.18 M NaCl/10 mM phosphate,