Large-scale proteomic analysis of membrane proteins

Proteomic analysis of membrane proteins is a promising approach for the identification of novel drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solublization of membrane proteins are encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Frequently, unknown proteins are identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict the presence of transmembrane domains. This review also presents these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.     

Large-scale proteomic analysis of membrane proteins

Proteomic analysis of membrane proteins is a promising approach for the identification of novel drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solublization of membrane proteins are encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Frequently, unknown proteins are identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict the presence of transmembrane domains. This review also presents these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.     

A method for the comprehensive proteomic analysis of membrane proteins

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.     

Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry

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.     

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.     

Differential analysis of membrane proteins in mouse fore- and hindbrain using a label-free approach

The ability to quantitatively compare protein levels across different regions of the brain to identify disease mechanisms remains a fundamental research challenge. It requires both a robust method to efficiently isolate proteins from small amounts of tissue and a differential technique that provides a sensitive and comprehensive analysis of these proteins. Here, we describe a proteomic approach for the quantitative mapping of membrane proteins between mouse fore- and hindbrain regions. The approach focuses primarily on a recently developed method for the fractionation of membranes and on-membrane protein digestion, but incorporates off-line SCX-fractionation of the peptide mixture and nano-LC-MS/MS analysis using an LTQ-FT-ICR instrument as part of the analytical method. Comparison of mass spectral peak intensities between samples, mapping of peaks to peptides and protein sequences, and statistical analysis were performed using in-house differential analysis software (DAS). In total, 1213 proteins were identified and 967 were quantified; 81% of the identified proteins were known membrane proteins and 38% of the protein sequences were predicted to contain transmembrane helices. Although this paper focuses primarily on characterizing the efficiency of this purification method from a typical sample set, for many of the quantified proteins such as glutamate receptors, GABA receptors, calcium channel subunits, and ATPases, the observed ratios of protein abundance were in good agreement with the known mRNA expression levels and/or intensities of immunostaining in rostral and caudal regions of murine brain. This suggests that the approach would be well-suited for incorporation in more rigorous, larger scale quantitative analysis designed to achieve biological significance.     

Chromatographic benefits of elevated temperature for the proteomic analysis of membrane proteins

Integral membrane proteins (IMPs) perform crucial cellular functions and are the primary targets for most pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains and their intimate association with lipids make them difficult to handle. Numerous proteomic platforms that include LC separations have been reported for the high-throughput profiling of complex protein samples. However, there are still many challenges to overcome for proteomic analyses of IMPs, especially as compared to their soluble counterparts. In particular, considerations for the technical challenges associated with chromatographic separations are just beginning to be investigated. Here, we review the benefits of using elevated temperatures during LC for the proteomic analysis of complex membrane protein samples.     

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

Background  

The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids.     

膜整合蛋白质的“鸟枪法”蛋白质组学分析策略

疾病状态下生物膜表面蛋白质分子标记的表达量和修饰状态会发生改变。但由于其低丰度和不易溶解等特性,制约了膜蛋白质组学的研究,同时也制约了相关药物靶标的设计。近年来,为克服这些困难,学者们提出了"鸟枪法"的膜蛋白质组学研究策略。基于此,本文论述了"鸟枪法"的蛋白质组学分析的基本过程及其后续的部分改进工作。随着新的策略不断被采用,更多膜蛋白质的拓扑学特征和功能的相关研究一定会走上新的台阶。     

Generic workflow for quality assessment of quantitative label-free LC-MS analysis

As high-resolution instruments are becoming standard in proteomics laboratories, label-free quantification using precursor measurements is becoming a viable option, and is consequently rapidly gaining popularity. Several software solutions have been presented for label-free analysis, but to our knowledge no conclusive studies regarding the sensitivity and reliability of each step of the analysis procedure has been described. Here, we use real complex samples to assess the reliability of label-free quantification using four different software solutions. A generic approach to quality test quantitative label-free LC-MS is introduced. Measures for evaluation are defined for feature detection, alignment and quantification. All steps of the analysis could be considered adequately performed by the utilized software solutions, although differences and possibilities for improvement could be identified. The described method provides an effective testing procedure, which can help the user to quickly pinpoint where in the workflow changes are needed.     

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer "sandwich" gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.     

Solid phase extraction of the zwitterionic detergent chaps

Multiple techniques for solid phase adsorption of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were evaluated. Both the porous polystyrene divinylbenzene matrices (BioBeads SMTM) and Extracti GelTM D reduced CHAPS to significantly below its critical micellar concentration while Extracti-GelTM removed CHAPS to below detectable limits. Bio-Bead extraction of CHAPS correlated with the surface area of the bead type. SM-16 beads, with the largest effective surface area, removed nearly 97% of the detergent. For a given amount of detergent and mass of Bio-Beads, the ratio of sample to total bead volume significantly affected CHAPS adsorption. Total protein recovery with the Extracti-GelTM was approximately 97%. Protein recovery in the samples treated with Bio-Beads varied from 56-95%. Chromatographic rather than batch processing yielded optimum recoveries. CHAPS can be effectively removed from dilute protein solutions by solid phase adsorption and this technique offers significant advantages over standard dialysis or gel filtration methods.     

Comprehensive proteomic analysis of Shigella flexneri 2a membrane proteins

Shigella flexneri is the causative agent of most shigellosis cases in developing countries. We used different proteolytic enzymes to selectively shave the protruding proteins on the surface of purified bacterial membrane sheets or vesicles, and recovered peptides were subsequently identified using 2-D LC-MS/MS. As a result, a total of 666 proteins were unambiguously assigned, including 159 integral membrane proteins, 35 outer membrane proteins and 114 proteins previously annotated as hypothetical. The former had an average grand average hydrophobicity score of 0.362 and were predicted to separate within a pH range of 4.1-10.6 with molecular mass 8-148 kDa, which represents the largest validated set of integral membrane proteins in this organism to date. A functional classification revealed that a large proportion of the identified proteins were involved in cell envelope biogenesis and energy production and conversion. For the first time, this work provides a global view of the S. flexneri 2a membrane subproteome.     

MS-BID: a Java package for label-free LC-MS-based comparative proteomic analysis

MS-BID (MS Biomarker Discovery Platform) is an integrative computational pipeline for biomarker discovery using LC-MS-based comparative proteomic analysis. This platform consists of several computational tools for: (i) detecting peptides in the collected patterns; (ii) matching detected peptides across a number of LC-MS datasets and (iii) selecting discriminatory peptides between classes of samples. AVAILABILITY: MS-BID source codes, binaries and documentations are freely available under LGPL from http://tools.proteomecenter.org/msBID.php.     

Nonionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent

The UV-visible, circular dichroism (CD), and resonance Raman (RR) spectra of the wild type yeast iso-1-cytochrome c (WT) and its mutant F82H in which phenylalanine-82 (Phe-82) is substituted with His are measured and compared for oxidized and reduced forms. The CD spectra in the intrinsic and Soret spectral region, as well as RR spectra in high, middle, and low frequency regions, are discussed. From the analysis of the spectra, it is determined that in the oxidized F82H the two axial ligands to the heme iron are His-18 and His-82 whereas in the reduced form the sixth ligand switches from His-82 to Met-80 providing the coordination geometry similar to that of WT. Based on the spectroscopic data, the conclusion is that the porphyrin macrocycle is less distorted in the oxidized F82H compared to the oxidized WT. Similar distortions are present in the reduced form of the proteins. Frequency shifts of Raman bands, as well as the decrease of the alpha-helix content in the CD spectra, indicate more open conformation of the protein around the heme.     

Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins

Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein‐detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the PDC, knowledge of these boundaries under different chemical conditions provides a foundation to rationally design crystallization screens. We have carried out dye‐based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents to generate a significant amount of chemically diverse phase boundary data. The resulting curves were used to guide the formulation of a 1536‐cocktail crystallization screen for membrane proteins. We are making both the experimentally derived phase boundary data and the 1536 membrane screen available through the high‐throughput crystallization facility located at the Hauptman‐Woodward Institute. The phase boundary data have been packaged into an interactive Excel spreadsheet that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. The 1536 membrane screen has been applied to 12 membrane proteins of unknown structures supplied by the structural genomics and structural biology communities, with crystallization leads for 10/12 samples and verification of one crystal using X‐ray diffraction.     

Quantitative proteomic analysis of membrane proteins involved in astroglial differentiation of neural stem cells by SILAC labeling coupled with LC-MS/MS

Membrane proteins play a critical role in the process of neural stem cell self-renewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the self-renewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trap-Orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation.     

Optimized proteomic analysis on gels of cell-cell adhering junctional membrane proteins

A high level of structural organization of functional membrane domains in very narrow regions of a plasma membrane is crucial for the functions of plasma membranes and various other cellular functions. Conventional proteomic analyses are based on total soluble cellular proteins. Thus, because of insolubility problems, they have major drawbacks for use in analyses of low-abundance proteins enriched in very limited and specific areas of cells, as well as in analyses of the membrane proteins in two-dimensional gels. We optimized proteomic analyses of cell-cell adhering junctional membrane proteins on gels. First, we increased the purity of cell-cell junctions, which are very limited and specific areas for cell-cell adhesion, from hepatic bile canaliculi. We then enriched junctional membrane proteins via a guanidine treatment; these became selectively detectable on two- dimensionally electrophoresed gels after treatment with an extremely high concentration of NP-40. The framework of major junctional integral membrane proteins was shown on gels. These included six novel junctional membrane proteins of type I, type II, and tetraspanin, which were identified by mass spectrometry and by a database sequence homology search, as well as 12 previously identified junctional membrane proteins, such as cadherins and claudins.     

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.