Quantitative proteomic analysis of the adipocyte plasma membrane

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.     

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.     

Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane

Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.     

Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.     

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.     

Functional properties of ryanodine receptors from rat dorsal root ganglia

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (Bmax) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (Kd) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+-dependent and displayed the classical long-lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+-dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.     

Evaluation of two cell surface modification methods for proteomic analysis of plasma membrane from isolated mouse hepatocytes

The hepatocyte is a highly polarized cell with a heterogeneous distribution of plasma-membrane (PM) proteins. To reduce the complexity of the proteome of liver tissue and give a comprehensive profile of hepatocyte PM, two PM purification methods based on cell surface modification, named the biotin-avidin (BA) and cationic silica-polyanion (CSP) strategies were evaluated and compared with the traditional cell fractionation method to prepare highly enriched PM from freshly isolated C57 mouse hepatocytes. Employing different principles for PM modification, both methods were effective in the isolation of general and purified PM fraction. The CSP strategy showed better yield for the PM purification from freshly isolated hepatocytes. 189 non-redundant proteins were identified, including 49 from the BA method and 185 from CSP strategy. Many known and novel PM-associated proteins were also found. Our evaluation here should give implications for PM preparation from other freshly isolated tissue-derived cells. The hepatocyte PM proteins identified here should be taken as a references for the PM-related functional and diseases research.     

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.     

Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.     

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

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.     

Enrichment of right-side-out Trypanosoma cruzi plasma membrane vesicles

A simple method to prepare a high yield of Trypanosoma cruzi plasma membrane vesicles (PMV) from epimastigotes and metacyclic trypomastigotes is described. The method may be applicable to other protozoa. Solid-phase immunoassay to bind surface T. cruzi epitopes showed that this preparation was enriched with 80-82% PMV and that most of these were right-side out (81-92%). The method was based on the extraction of extrinsic proteins and subpellicular tubules with mild high and low ionic strength buffers without detergents (pH 7.4) and on the differential centrifugation of PMV based on their specific density (1.049 g/ml, 4 degrees C). Transmission electron microscopy of PMV pellets showed a heterogeneous population of vesicles without other significant cytoskeletal contaminants. T. cruzi PMV were also enriched with an ouabain- and oligomycin-insensitive magnesium-ATPase and contained an adenylyl cyclase, preserved for at least 3 months at -70 degrees C in storage buffer. Measurements of the [14C]-dextran and the 3H2O space indicated that T. cruzi PMV were not sealed, explaining why Lubrol PX and NaF failed to stimulate the adenylyl cyclase activity further and why T. cruzi PMV were unable to concentrate 86Rb in flow dialysis assays. No detectable DNA and RNA was found. The preparation was not capable of removing 51Cr or [3H]glucosamine from live L6 myoblast surfaces in physiologic conditions and acid phosphatase was extracted by this method. The contaminating fraction (18-20% by immunoassay) consisted of endoplasmic reticulum membranes with NADH oxidase activity and of kinetoplast membranes with cytochrome c oxidase and oligomycin sensitive magnesium-ATPase activity. The biologically active T. cruzi PMV retained the ability of living forms to trigger the alternate pathway of complement by releasing the Bb activation fragment from human Factor B.     

真核细胞质膜蛋白质组研究进展

细胞膜（质膜）蛋白质是细胞的“门铃”与“门户”,是许多药物的作用靶标。细胞质膜蛋白质组的研究正成为蛋白质组研究的热点,这方面的研究有利于具有重要功能的低丰度蛋白质的发掘,为药物研发和疾病的诊断提供靶体与标记蛋白质。然而,质膜蛋白质组的研究在强疏水性跨膜蛋白质和低丰度膜蛋白质的分离和鉴定上遇到了方法学的挑战。本文对质膜及其微区的纯化、质膜蛋白质组的分离与鉴定、生物信息学,以及亚细胞定位研究的近期进展作扼要介绍。     

NADH-specific dehydrogenase from onion root plasma membrane: purification and characterization

Summary Plasma membranes isolated from onion roots by twophase partition contain at least two different NAD(P)H-dehydrogenases. A 27 kDa electron transport protein oxidises both NADH and NADPH and exhibits maximal activity with quinones as electron acceptors. A distinct 31 kDa dehydrogenase is specific for NADH as donor and shows maximal activity with ferricyanide. This novel enzyme is responsible for most NADH-ferricyanide oxidoreductase activity of solubilized onion root plasma membranes and exhibits properties different to other purified NAD(P)H-dehydrogenases.Abbreviations DES diethylstilbestrol - FeCN potassium ferricyanide - NBT nitroblue tetrazolium - PHMB p-hydroxymercuribenzoate - PMSF phenylmethylsulfonylfluoride - PTA phosphotungstic acid - SHAM salicylhydroxamic acid - TTFA thenoyltrifluoroacetone     

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.     

Prenatal development of the rat dorsal root ganglia

Summary This study describes three-dimensional aspects of the development and pseudo-unipolarization of neuroblasts and the maturation of satellite cells in prenatal rat dorsal root ganglia, using scanning electron microscopy, after removal of extracellular connective tissue components by trypsin digestion and HC1 hydrolysis.At 14 days of gestation, the vast majority of neurons are spindle-shaped or bipolar and only 3% are unipolar, while at 16 and 18 days this percentage has increased to 30% and 91%, respectively. The initial portions of the central and peripheral neuronal processes gradually approach each other and form a common initial portion. Finally, the cytoplasm of this common initial portion becomes thinner and elongates to form the stem process of the mature cell.Satellite cells are present from the beginning of the period studied, but intricate networks of branching satellite cell processes only develop after about day 17.