A method for the comprehensive proteomic analysis of membrane proteins (original) (raw)
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A Simple and Effective Method to Analyze Membrane Proteins by SDS-PAGE and MALDI Mass Spectrometry
2010
Background/Aim: Identification and characterization of membrane proteins is a crucial challenge in proteomics research. Thus, we designed a novel method to prepare proteins possessing extensive hydrophobic stretches for mass spectrometry studies, without sacrificing other classes of proteins. Materials and Methods: This method uses sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and relies solely on a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) instrument, the most common and easiest to use mass spectrometer. Results: Using this analytical procedure, a significant number of hydrophobic peptides were recovered, with no reduction in overall sequence coverage and with a good identification of transmembrane proteins sequence. Applying this method to the systematic identification of proteins located in lipid rafts, up to 47% of identified proteins were obtained with an improvement of sequence coverage. Conclusion: The procedure presented here is suitable for both identifying purified hydrophobic proteins and systematically investigating hydrophobic protein mixtures. It can be easily applied even in non-dedicated laboratories, such as those mostly devoted to clinical chemistry. About 30% of all proteins in nature are either membrane associated (MA, mostly hydrophilic), or integral membrane proteins (IMP, mostly hydrophobic). Compared with MAs, IMPs are fully embedded into the phospholipid bilayer and play a cardinal role in cell-to-cell interactions, substrate transport, and signal transduction (1). Despite the great interest in characterizing membrane proteins, their strong hydrophobicity, mainly due to α-helical bundles, is a hurdle for most common proteomic approaches, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography, and mass spectrometry (2, 3). As a consequence, membrane proteins are usually poorly represented in proteomic analyses (4). Purification of IMPs by gel electrophoresis presents a significant problem, since their hydrophobic nature makes them prone to precipitation, or to be trapped in the gel's mesh, mainly when the buffer pH reaches their isoelectric point, or in the absence of appropriate detergents and/or reducing agents. Since both trapping and precipitation depend on folding of hydrophobic domains, it has been proposed that their proteolytic hydrolysis might be an appropriate solution; however, this could lead to the production of highly hydrophobic peptides, no less difficult to recover than whole IMPs themselves (5). The aim of this study was to set up a simple, effective, and reliable method (RM) to analyze MPs in a non-specialized proteomics laboratory setting. For benchmarking our work, we compared our method (RM) with a more conventional method for protein preparation in mass spectrometric analysis (CM) (6). To illustrate the favorable characteristics of RM, we characterized mouse band 3 (an IMP), mouse βactin (an MA), the rat eight-transmembrane-strand MLC1 (megalencephalic leukoencephalopathy), and malaria parasite Plasmodium berghei pbSEP1, an IMP with one transmembrane portion. Finally, we used both RM and CM to identify rat lipid-raft membrane proteins and compared the respective effectiveness of the two methods. Materials and Methods Reagents and chemicals. SDS and ammonium bicarbonate (NH 4 HCO 3) were obtained from INC Biochemicals (Milwaukee, WI, USA). Iodoacetamide (IAA) and cyanogen bromide (CNBr) were purchased from Fluka (Milwaukee, WI, USA), 1,4-dithiothreitol (DTT) and α-cyano-4-hydroxycinnamic acid matrix (CHCA) from
Integral membrane proteins: bottom-up, top-down and structural proteomics
Expert Review of Proteomics, 2017
Introduction: Integral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review. Areas covered and literature search strategy: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput topdown proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing critical in-vivo information. Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized.
Journal of Proteome Research, 2002
An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.
Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease "shaving" of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original "hppK" method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1-16 transmembrane segments. This method allowed us to identify 13 "missing proteins"proteins with no previous evidence on protein level. Biological significance: Integral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called "missing proteins".
Comparative and Functional Genomics, 2002
Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural prot...
A robust purification strategy to accelerate membrane proteomics
Methods, 2007
The preparation of large quantities of puriWed membrane proteins for structural studies presents signiWcant diYculties. Central among these are the frequent toxicity associated with over-expressing membrane targets and the diYculty associated with identifying the appropriate detergents for their solubilization and puriWcation. To begin addressing these challenges, and lay the groundwork for membrane structural genomics eVorts, we have developed a robust strategy for the expression and puriWcation of large numbers of prokaryotic membrane proteins. Our approach rapidly identiWes highly expressed targets and greatly simpliWes their solubilization and puriWcation. In this review, speciWc, hands-on protocols are provided for the expression and puriWcation of CorA magnesium transporters. These methods form the basis for the expression and puriWcation of many other membrane proteins, as discussed.
QARIP: a web server for quantitative proteomic analysis of regulated intramembrane proteolysis
Nucleic Acids Research, 2013
Regulated intramembrane proteolysis (RIP) is a critical mechanism for intercellular communication and regulates the function of membrane proteins through sequential proteolysis. RIP typically starts with ectodomain shedding of membrane proteins by extracellular membrane-bound proteases followed by intramembrane proteolysis of the resulting membrane-tethered fragment. However, for the majority of RIP proteases the corresponding substrates and thus, their functions, remain unknown. Proteome-wide identification of RIP protease substrates is possible by mass spectrometry-based quantitative comparison of RIP substrates or their cleavage products between different biological states. However, this requires quantification of peptides from only the ectodomain or cytoplasmic domain. Current analysis software does not allow matching peptides to either domain. Here we present the QARIP (Quantitative Analysis of Regulated Intramembrane Proteolysis) web server which matches identified peptides to the protein transmembrane topology. QARIP allows determination of quantitative ratios separately for the topological domains (cytoplasmic, ectodomain) of a given protein and is thus a powerful tool for quality control, improvement of quantitative ratios and identification of novel substrates in proteomic RIP datasets. To our knowledge, the QARIP web server is the first tool directly addressing the phenomenon of RIP. The web server is available at http://webclu. bio.wzw.tum.de/qarip/. This website is free and open to all users and there is no login requirement.
Mass spectrometry accelerates membrane protein analysis
Trends in Biochemical Sciences, 2011
Cellular membranes are composed of proteins and glyco-and phospholipids and play an indispensible role in maintaining cellular integrity and homeostasis by physically restricting biochemical processes within cells and providing protection. Membrane proteins perform many essential functions, which include operating as transporters, adhesion-anchors, receptors, and enzymes. Recent advancements in proteomic mass spectrometry have resulted in substantial progress towards the determination of the plasma membrane (PM) proteome, resolution of membrane protein topology, establishment of numerous receptor protein complexes, identification of ligand-receptor pairs, and the elucidation of signaling networks originating at the PM. Here we discuss the recent accelerated success of discovery-based proteomic pipelines for the establishment of a complete membrane proteome.
Methods, 2011
Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrosprayionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gasphase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent highresolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent posttranslational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of posttranscriptional and post-translational diversity for enhanced understanding beyond genomic translation.
PROTEOMICS, 2004
A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (mLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by mLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single mLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.