Membrane proteins represent a valuable source of potential drug targets due to their intimate involvement in a wide variety of disease states, including diabetes, cancer and neurological disorders. Defining the proteome of these often rare amphipathic molecules can be accomplished by exploiting the highly accurate and sensitive nature of mass spectrometry (MS). Technical advances have enabled MS to become a valuable tool for detailed mechanistic investigations into membrane proteins of unknown and known structure. The transfer of MS-screening strategies that have already been successfully used to identify interactions between soluble proteins and potential ligands, should allow the identification of drug candidates for membrane proteins in the near future.
[Show abstract][Hide abstract] ABSTRACT: This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.
[Show abstract][Hide abstract] ABSTRACT: Membrane proteins play an important role in almost all cell activities. However, the characterization of the structure of membrane proteins in lipid bilayers is still at the frontier of structural biology. While 30-40% of all proteins are situated at or in membranes, yet less than 1% of the known protein structures are of membrane proteins. The complexity and delicacy of membrane-protein systems impedes the application of standard methods of protein study, such as X-ray crystallography and NMR. Furthermore, these techniques are mainly aimed at short-range structural information, and seem to be not very useful for the study of long-range interactions, for instance in the case of protein assemblies. These factors urge to find other biophysical approaches to study proteins incorporated into lipid bilayers. A successful alternative is Förster (or fluorescence) resonance energy transfer (FRET) spectroscopy in combination with site-directed labeling with fluorescent probes. This technique provides distance information within a range of 10-100 Å, which is sufficient to study the structure of membrane proteins and their complexes. The crucial point in the extraction of structural information from FRET data is an advanced and robust data analysis. This work is devoted to the development of such methods for analysis of fluorescence data, based on simulation modeling, global analysis and artificial neural networks. Especially the advances and problems of the simulation-based fitting (SBF) approach to fluorescence data analysis are considered. The methodologies of global analysis and SBF are applied to obtain information about the position, aggregation and structure of M13 major coat protein in DOPC :DOPG vesicles. The resulting physical parameters, that describe the embedment and orientation of the protein in the membrane, such as protein-protein aggregation, protein depth, tilt angle, and tilt direction, are in good accordance with previously reported values. Based on the FRET data, it was found that M13 major coat protein (having 50 amino acid residues) in its bilayer conformation could be described as a single a -helix between amino acid positions 10-46. Additional work was performed on the methodological aspects of improving the SBF data analysis technique. Here it is proposed to use an artificial neural network to speed up the parameter identification and to make the process of fitting less sensitive to noise. The main idea of this method is the substitution of a time-consuming simulation model by an artificial neural network, specifically a multi-layer perceptron. The method results in a speeding up of the simulation by about a factor of 10 <sup>4</sup> for the developed FRET model.
[Show abstract][Hide abstract] ABSTRACT: Analyses of proteins from a number of proteomic studies of cell membranes have demonstrated that a significant component of the identified proteins is not predicted to contain transmembrane regions. The presence of such proteins may arise as a result of contamination of the membrane preparations or through real associations. Our aim was to identify integral proteins as well as those that are intimately associated with the microsomal membranes of K562 cells. Isolated membranes were treated under conditions reported to remove noncovalently associated 'peripheral' proteins and the residual proteins were SDS-PAGE-separated and analyzed by LC-MS/MS. Tandem lectin affinity was also examined as a complementary approach for the enrichment of membrane glycoproteins. Approximately 41% of the isolated proteins were assigned as membrane proteins based on the presence of transmembrane regions or covalent post-translational modifications that could account for membrane association. Collectively, these results indicate that there is a significant component of non integral proteins that appear to be as closely associated with membranes as integral elements.
Journal of Proteome Research 05/2008; 7(4):1572-83. DOI:10.1021/pr070509u · 4.25 Impact Factor
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