Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

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.     

Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.     

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.     

大鼠背根神经节细胞质膜的双水相法纯化及其蛋白质组学研究

大鼠背根神经节(dorsal root ganglion, DRG)细胞是一种初级感觉神经元,能传导触觉、痛觉、温觉等神经冲动．为了对少量的DRG组织细胞进行质膜蛋白质组学分析,综合利用差速离心与双水相相结合的方法富集DRG质膜．然后通过SDS-PAGE、CapLC-MS/MS和生物信息学方法对其中的蛋白质进行鉴定和分析．Western blotting图谱扫描后经过Quantity One软件分析,双水相纯化后的质膜与差速离心后得到的粗质膜相比相对浓度增加了2.3倍,与匀浆液相比增加了15倍． 经过大鼠IPI数据库以及相关文献检索, 有729个蛋白质得到鉴定, 其中547个蛋白质具有GO (gene ontology)注释信息,有159 (21.8 %)个蛋白质定位在质膜上．通过对大鼠DRG质膜的蛋白质组学研究,得到了大鼠DRG的质膜蛋白质的分析数据,且提供了一种适用于少量样品的蛋白质组学的分析路线．     

Proteomic mapping of brain plasma membrane proteins

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.     

A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

Isolation and sequence of tryptic peptides from the proton-pumping ATPase of the oat plasma membrane

In crude extracts of plant tissue, the M_r = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4°C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases.     

大鼠海马细胞质膜的分离及其蛋白质组学分析

运用差速离心和连续蔗糖密度梯度离心的方法分离纯化成年大鼠海马组织细胞质膜并用Western blotting对质膜样品进行检测.结果表明,在蔗糖密度梯度离心介质中,膜片主要集中于蔗糖浓度为32%~42%的区段(密度约1.13~1.18),其次是20%~25% (密度约1.08~1.10)的区段.39%(密度约1.17)层面上质膜浓度和纯度最高.一维SDS-PAGE分离和CapLC-MS/MS分析后,通过数据库搜寻从大鼠海马组织细胞质膜样品中共鉴定出135种蛋白质, 其中质膜蛋白和与膜相关的蛋白质共70种(占51.9%), 主要包括Na⁺/K⁺-ATPase、Glutamate/aspartate transporter、Lipophilin、GLAST 1a等. 为研究海马组织细胞的功能和大鼠海马组织细胞质膜蛋白质数据库的建立积累了有价值的资料.     

Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis

In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC‐MS‐based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two‐aqueous‐phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H⁺‐ATPases, ATP‐binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 ( http://proteomecentral.proteomexchange.org/dataset/PXD000837 ).     

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.     

A dynamic plasma membrane proteome analysis of alcohol-induced liver cirrhosis

ABSTRACT: Alcohol-induced injury has become one of the major causes for liver cirrhosis. However, the molecular mechanisms of ethanol-induced injury are not fully understood. To this end, we performed a dynamic plasma membrane proteomic research on rat model. A rat model from hepatitis to liver cirrhosis was developed. Plasma membrane from liver tissue with liver fibrosis stage of 2 and 4 (S2 and S4) was purified by sucrose density gradient centrifugation. Its purification was verified by western blotting. Proteins from plasma membrane were separated by two-dimensional electrophoresis (2DE) and differentially expressed proteins were identified by tandem mass spectrometry. 16 consistent differentially expressed proteins from S2 to S4 were identified by mass spectrometry. The expression of differentially expressed proteins annexin A6 and annexin A3 were verified by western blotting, and annexin A3 was futher verified by immunohistochemistry. Our research suggests a possible mechanism by which ethanol alters protein expression to enhance the liver fibrosis progression. These differentially expressed proteins might be new drug targets for treating alcoholic liver cirrhosis.     

大鼠背根神经节细胞膜蛋白的提取及双向凝胶电泳分离

为了开展大鼠背根神经节(DRG)细胞质膜蛋白质组学研究,取成年大鼠的背根神经节,用胰蛋白酶和胶原酶等消化处理后经密度梯度离心分离DRG细胞质膜;用裂解液裂解提取膜蛋白并通过双向凝胶电泳将膜蛋白分离.扫描凝胶图谱后进行图像分析,结果表明DRG细胞膜蛋白得到了有效的提取和分离.双向凝胶电泳图谱的建立为进一步进行DRG细胞质膜蛋白质组学的研究提供了重要的基础.     

Isolation and partial characterization of the plasma membrane from human spermatozoa

Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM). Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions. The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation. A single membrane population was resolved at the 1.0 M sucrose interface. Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures. Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5' nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase). Analytical lipid analysis of the isolated membranes revealed a 26-fold enrichment in the distribution of cholesterol, an 11-fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 0.83. Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells. These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM.     

Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry

To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning beta-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally unclassified despite being predicted to be membrane proteins.     

High-throughput analysis of rat liver plasma membrane proteome by a nonelectrophoretic in-gel tryptic digestion coupled with mass spectrometry identification

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.     

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.