Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Increasing the resolution of polyacrylamide gel electrophoresis by varying the degree of gel crosslinking

Resolution of polyacrylamide gel electrophoresis may be substantially improved by taking advantage of the gel sieving effects of varying concentrations of bisacrylamide crosslinker. A dilution procedure is described which permits simultaneous variation of both total acrylamide concentration and percent crosslinking within a single linear regression analysis.This work was supported by NSF Grant 10584 and NIH Grant 23504.     

Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.     

Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.     

Proteomic analysis of Candida magnoliae strains by two-dimensional gel electrophoresis and mass spectrometry

Candida magnoliae which has been newly isolated from honey comb is an osmotolerant yeast to produce erythritol as a major product. Erythritol is a noncariogenic, low calorie sweetener and safe for diabetics. Strain development by chemical mutation to obtain the improved erythritol yield and productivity relative to the parental strain made it necessary to elucidate the physiological differences between the wild and mutant strains. Proteomic analyses of C. magnoliae wild and mutant strains with two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry were carried out to identify intracellular proteins and to estimate the effects of newly characterized metabolic enzymes on the yeast cell growth and erythritol production. Most of the molecular mass of intracellular proteins were distributed in the range of pI 4-8 and molecular mass of approximately 130 kDa. Six out of nine protein spots expressed at different levels between the wild and mutant strains were analyzed with nanoelectrospray tandem mass spectrometry and identified by comparing amino acid sequences with the National Center for Biotechnology Information and Saccharomyces Genome Databases. Except for Ygr086cp, these proteins were believed to be the metabolic enzymes involved in the citric acid cycle (citrate synthase, succinyl-CoA ligase and fumarase) and the glycolysis pathway (pyruvate decarboxylase and enolase). Up-regulated enzymes in the citric acid cycle could explain high growth of the C. magnoliae mutant strain owing to the increased NADH and ATP formation. Down-regulated enolase and up-regulated fumarase in the mutant strain seemed to play a role in the improved bioconversion of erythrose-4-phosphate to erythritol compared with the wild strain.     

盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验的几点改进

对盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验提出了几点改进,以满足本科生实验的要求。实验主要比较和分析了两种封胶方法（原胶布封胶与改进的琼脂糖封胶）和两种染色方法（原考马斯亮蓝染色法与改进的考马斯亮蓝染色法）对凝胶分离血清蛋白实验的影响。结果显示,改进的盘状聚丙烯酰胺凝胶电泳法是一种灵敏、快速、简便、安全、分辨率高的实验方法。结论：改进的盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验非常适合本科生实验。     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.     

Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry

Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.     

Genetic analysis of gliadin components in winter wheat using two-dimensional polyacrylamide gel electrophoresis

Summary Blocks of gliadin components found both in a number of varieties and in single F₂ grains of winter wheat intervarietal hybrids have been studied by two-dimensional electrophoresis combining electrophoresis in acidic aluminium-lactate buffer (pH3.1) and SDS-electrophoresis. Gliadin components (spots) have been shown to be inherited as linked groups (blocks), codominantly and in accordance with a gene dosage in triploid endosperm. Blocks include components differing in their electrophoretic mobility and molecular weight. Some allelic variants of blocks differ only in presence of few additional components or in the electrophoretic mobility of components with similar molecular weights; other variants may contain no similar components. Apparently, in the course of evolution, mutations in individual genes of gliadin-coding loci and processes changing the number of expressing genes and the sizes of their structural part occurred.     

The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis

The glycans of glycoconjugates mediate numerous important biological processes. Their separation and structural determination present considerable difficulties because of the small quantities that are available from biological sources and the inherent difficulty of analyzing the wide variety of complex structures that exist. A method for the analysis of reducing saccharides by PAGE that uses specific fluorophore labeling and is simple, rapid, sensitive, and readily available to biological researchers, has been developed. The method is known acronimically either as PAGEFS (PAGE of Fluorophore-labeled Saccharides) or in one commercial format as FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). In the PAGEFS method, saccharides having an aldehydic reducing end group are labeled quantitatively with a fluorophore and then separated with high resolution by PAGE. Two fluorophores, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS) and 2-aminoacridone (AMAC), have been used to enable the separation of a variety of saccharide positional isomers, anomers, and epimers. Subpicomolar quantities of individual saccharides can be detected using a sensitive imaging system. Mixtures of oligosaccharides obtained by enzymatic cleavage from glycoproteins can be labeled and electrophoresed to yield an oligosaccharide profile of each protein. AMAC can be used to distinguish unequivocally between acidic and neutral oligosaccharides. Methods for obtaining saccharide sequence information from purified oligosaccharides have been developed using enzymatic degradation. Other applications and the potential of the system are described.     

Resolution ofFusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence homology comparison in protein data base

Proteins fromFusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data.     

Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry

Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5-10), followed by MALDI-TOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis.     

苏云金杆菌4.0718菌株杀虫晶体蛋白的双向电泳飞行时间质谱分析

根据苏云金杆菌4.0718菌株杀虫晶体蛋白的特性,对裂解液组成、上样量、聚焦时间等相关技术进行了比较研究和条件优化,首次获得苏云金杆菌杀虫晶体蛋白双向电泳图谱,并对部分蛋白质点进行胰酶酶解,基质辅助激光解吸电离飞行时间质谱（matrixassisted laser desorption/ionization time of flight mass spectrometry,MALDITOFMS）测定肽质量指纹图谱,Mascot软件查询SwissProt数据库,最终鉴定出苏云金杆菌4.0718菌株伴孢晶体中所含的Cry1Ac和Cry2Aa蛋白,其精确分子量分别为134160Da和71097Da。     

Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.