Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methods

Snake venoms contain a large number of biologically active substances and the venom components are very useful for pharmaceutical applications. Our goal is to separate and identify components of snake venoms in ten snake species from the Elapidae and Viperidae families using multidimensional chromatographic methods. The multidimensional chromatographic methods include reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip, two-dimensional electrophoresis (2-DE), and mass spectrometry. The venoms of eight snake species demonstrated major differences in hydrophobicity, molecular weight separations, and 2-DE protein distribution patterns. The 2-DE images showed major differences between families, within each family and even between the same species. Venoms of the Elapidae family showed many basic proteins with a wide range of molecular weights, while venoms of the Viperidae family showed wide ranges of pI and molecular weights, especially for Trimeresurus sp. The multidimensional chromatographic methods revealed specific differences in venom proteins intra-species as well as between species and families. We have isolated and identified proteins that may be unique for each species for further studies in the proteome of snake venoms and their potentially use in the pharmaceutical applications.     

分泌蛋白质组研究进展

分泌蛋白质组是指组织、细胞等分泌的全部蛋白质。分泌蛋白主要分布于体液和细胞质胞浆中,参与许多重要的生命过程。分泌蛋白质组的研究主要涉及分泌蛋白的制备、多维色谱或二维凝胶电泳分离、质谱鉴定及生物信息学分析等。本文主要介绍了分泌蛋白的合成途径、蛋白质组技术在分泌蛋白组研究中的应用、分泌蛋白组研究现状及存在的问题等。     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

Separation and characterization of basic barley seed proteins

Basic proteins in barley starchy endosperm from developing seeds were separated by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as partial extracts were analyzed. Edman degradation sequencing and immunological detection were performed after transfer of separated proteins onto membranes. Only one protein could be analyzed by N-terminal sequencing of blotted and separated proteins from the total extract. Fractionation of extracts was done using cation exchange chromatography, concanavalin A and heparin affinity chromatography. Internal sequences were determined after in-gel cleavage of proteins using trypsin or cyanogen bromide and separation of the fragments by reversed-phase chromatography or in a gel electrophoresis system for peptide separation. This resulted in a new protocol for obtaining internal sequences from proteins separated by 2-D electrophoresis. A total of 16 sequences, including nine internal sequences, were analyzed, permitting the identification of ten proteins, including five that appeared to have a blocked N-terminus. An additional protein was identified using immunological detection. Three protein sequences remained unidentified. Separated proteins were also analyzed with a glycan detection method.     

Column chromatographic prefractionation leads to the detection of 543 different gene products in human fetal brain

In a previous publication a large series of proteins were identified in fetal human brain by the use of two-dimensional electrophoresis (2-DE) with subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and MALDI-tandem time-of-flight (TOF/TOF) analysis. Further identification of many more different spots by traditional 2-DE without additional step such as narrow immobilized ph gradient (IPG) strips or prefractionation seems unlikely and we therefore decided to separate extracted brain proteins by ion-exchange chromatography using a TSK gel DEAE-5PW column followed by 2-DE of individual fractions and analysis by MALDI-TOF/TOF with LIFT technology in fetal brain of the early second trimester. About 1880 protein spots corresponding to 543 different gene products were identified. These proteins included housekeeping, signaling, cytoskeletal, metabolic, antioxidant, and neuron/synaptosomal specific proteins. Among these, 314 gene products (314/543, 57.8%), which have never been detected in traditional 2-DE of human fetal brain, were observed by this method. This updated map of fetal brain proteins may serve as data base and reference map for fetal brain proteins, and the methodology applied may be used as a valuable analytical tool for the basis of protein expressional studies in health and disease.     

Identification of human myocard proteins separated by two-dimensional electrophoresis.

N-Terminal sequencing, internal sequencing and amino acid analysis were used to identify twelve proteins of the human myocard two-dimensional gel electrophoresis (2-DE) pattern. Amino acid analysis was shown to be a powerful tool in addition to sequencing. The identification of a disease-associated N-terminally blocked protein by internal sequencing was not successful. The twelve identified proteins are the basis of a human myocard 2-DE database.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins

Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.     

Towards a two-dimensional database of common human proteins.

A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

Comparison of serum protein removal in haemodialysis therapy by partly protein-permeable haemodialysers

Partly protein-permeable haemodialysers were evaluated during haemodialysis therapy for removal of serum low-molecular-mass proteins (10,000-76,000) in patients with chronic renal failure. The six haemodialyser membranes used were cuproammonium rayon (CL-S12W), cellulose acetate (Duo-Flux HP and FF-22), ethylene-vinyl alcohol copolymer (KF-101-15), polyacrylonitrile (H12-2400S) and polymethyl methacrylate (BK2.0H). The analysis was carried out by gel permeation chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE). The removal of ten serum proteins per haemodialysis therapy was also carried out by the immunodiffusion method. The protein removal in each haemodialyser is qualitatively comparable to that obtained by SDS-PAGE and 2-DE. beta 2-Microglobulin in the haemodialysate obtained with BK2.0H was removed to a lesser extent than that from the other haemodialysers, which seems to be the reason for its adsorption in the BK2.0H haemodialyser, which contains a polymethyl methacrylate membrane. The amount of serum protein excretion during haemodiafiltration treatment using the partly protein-permeable haemodialysers decreased in the order KF-101-15C greater than BK2.0H greater than CL-S12W greater than Duo-Flux HP greater than FF-22 greater than H12-2400S.     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

Workshop on two-dimensional gel protein databases.

A workshop on two-dimensional gel electrophoresis (2-DE) protein database, organized by the Committee on Data for Science and Technology (CODATA) of the International Council of Scientific Unions Task Group on Biological Macromolecules, was held at the CODATA Secretariat in Paris on March 9, 1992. Eleven scientists from eight different countries represented various aspects of 2-DE analysis--namely, cellular protein database development and protein microsequencing methodologies. The purpose of the workshop was to explore means of integrating the rapidly expanding body of information on 2-DE resolved proteins from different laboratories. A major proposal emanating from the workshop was the establishment of an intermediary or "relational" 2-DE gel protein database. This intermediary database, which would catalogue pertinent information on 2-DE resolved proteins (experimental source, 2-DE loci, biological information, etc.) could be an adjunct to, and accessed through, the existing international protein sequence databanks. It would function as a pointer for researchers to the individual 2-DE protein databases where primary and more specialized 2-DE data would be housed.     

Protein changes in post mortem sea bass (Dicentrarchus labrax) muscle monitored by one- and two-dimensional gel electrophoresis

This study was devoted to the identification of specific peptides and proteins which can be used as indicators of freshness in fish. The post mortem evolution of protein patterns in farmed sea bass muscle was monitored by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) after 0, 2, 4, and 6 days cold storage. SDS-electrophoresis, of total proteins and proteins soluble in low-ionic-strength solutions, revealed the gradual disappearance of a protein band of 16 kDa immediately after fish death. 2-DE allowed the classification of fish samples according to post mortem time. Three spots of interest, which disappeared progressively, were identified on the 2-DE patterns. Further research is required to establish the identity of these polypeptides and to evaluate their expression and post mortem evolution in another fish species.     

Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non-2-DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained gel was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-gel tryptic digestion of each slice were individually subjected to capillary-column reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS). Interrogation of genomic databases with the resulting collision-induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2-DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a "targeted" ion approach was used to unambiguously identify known low-abundance plasma membrane proteins, using the membrane-associated A33 antigen, a gastrointestinal-specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an "inclusion" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated "nontargeted" approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.     

Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources

Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.     

Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry

We paper describe a mass spectrometric approach generally applicable for the rapid identification and characterization of proteins isolated by two-dimensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electrospray mass spectrometry employing a quadrupole-time of flight mass spectrometer was used for the direct identification of proteins from the peptide mixture generated from only one high resolution 2-DE gel without high performance liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this technique proved to be a powerful tool for the identification of proteins from femtomole amounts of materials. We applied the technique for the investigation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 36 protein spots were examined, from only one micropreparative Coomassie Brilliant Blue R-250 stained gel, after proteolytic digestion by matrix assisted laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully identified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databases. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The given technique and the protein data are the basis for construction of a database to compare normal and apoptosis-induced cells and, further, to enable fast screening of drug impact in apoptosis-associated processes.     

Study of human cerebrospinal fluid proteins by size exclusion-high performance liquid chromatography and two-dimensional gel electrophoresis

Cerebrospinal fluid (CSF) proteins were separated into three main fractions by size exclusion-high performance liquid chromatography (SE-HPLC). Subsequent analysis of each fraction by two-dimensional gel electrophoresis (2-DE) facilitated the detection of trace components in CSF and additionally provided more information about the native properties of various proteins. Certain proteins are present in a polymeric form and appear in the high molecular weight SE-HPLC fraction. In the middle molecular weight SE-HPLC fraction we found a CSF-specific transthyretin-related protein by immunoblotting with polyclonal antibodies to transthyretin. Possible interpolypeptide disulfide bonds of such polymeric proteins were studied using a nonreducing 2-DE system. This procedure revealed that all apolipoprotein E monomers in CSF, which are synthesized in astrocytes, are linked by disulfide bonds. In the CSF from a patient with clinically definite multiple sclerosis (MS), novel proteins appeared in the high molecular weight SE-HPLC fraction, which are obscured by other proteins if total CSF is analyzed.