The resolution of membrane proteins based upon size, charge, and hydrophobicity

Currently available systems for resolving membrane proteins are based only on size and charge differences. Recently, it has been shown that Triton-urea-acetic acid gels which separate proteins on the basis of charge, size and hydrophobicity are capable of resolving proteins differing only by the substitution of a single neutral amino acid. We have applied this new method to the resolution of bacterial envelope proteins. Conditions for optimal resolution of different bacterial envelope proteins were determined by electrophoresis through transverse urea and Triton X-100 gradient gels. We have also correlated the components resolved in this system with those resolved by classical sodium dodecyl sulfate-gel electrophoresis by using two-dimensional slab gels combining the two systems. Furthermore, envelope protein fractions from different species and strains of bacteria were compared to identify specific proteins. This system appears to be a promising method for investigating envelope proteins which are due to missense mutations.     

Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5-200 kDa. For the first-dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second-dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Targeting T cells against brain tumors with a bispecific ligand-antibody conjugate

Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two-dimensional gel electrophoresis, during which immobilized pH 3-10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two-dimensional gel electrophoresis, using for the first-dimensional separation immobilized pH gradient strips comprising the pH region of 6-11. The protein spots were analyzed by matrix-assisted laser desorption ionization-mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two-dimensional map.     

Restriction sites as identification tags for lymphocyte cDNAs

A cDNA library was prepared from BW 5147 murine lymphoma cells in lambda ecc III phage and randomly partitioned into 291 sectors, each with 800-1000 recombinant phage plaques. One sector was chosen for further characterization in terms of sensitivity to restriction endonuclease cutting. Aliquots of DNA preparations from this sector were treated with XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI before being used as templates in a cell-free expression system. The polypeptide products were separated by two-dimensional (2-D) gel electrophoresis and radiofluorographs of the gels were submitted to computer-aided image analysis. The matched patterns were inspected for the presence or absence of spots upon individual endonuclease treatments. Thereafter the results were integrated in a data matrix which served as a basis to construct "restriction tags" for all spots. These (restriction) tags are binary numbers termed "cut numbers" and are a representation of the set of recognition sequences which are (or are not) part of the coding sequence. From 493 sequences (visualized as 2-D gel spots), 12 were not cut by any of the nine enzymes, while 45 were cut by all of them. The percentages of sequences resistant to enzyme treatment ranged between 17% and 77% for NcoI and XhoI, respectively. The enzyme treatments led to the appearance of a certain portion of "new spots", probably products from truncated sequences. From 512 possible cut numbers, 136 were assigned to the 493 spots. Restriction tags are available to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.     

Alzheimer brain proteins investigated by two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension

Two-dimensional (2-D) gel electrophoresis with immobilized pH gradient 4-8 in the first dimension was applied in the analysis of Alzheimer's disease brain proteins. The silver-stained 2-D maps of extracts from the frontal cerebral cortex were examined. About 800 and 550 protein spots could be observed on the electrophoretograms from the total and buffer-soluble fractions, respectively. In comparing the gels, four protein spots could now be detected which had either been hitherto undetectable (one spot) or which were weaker (two spots) or stronger (one spot) in density (in the controls) [corrected].     

Proteins induced in Escherichia coli by benzoic acid

Proteins induced by benzoic acid in Escherichia coli were observed on two-dimensional electrophoretic gels (2-D gels). Cultures were grown in glucose-rich medium in the presence or absence of 20 mM benzoate at an external pH of 6.5, where the pH gradient (deltapH) is large and benzoate accumulates, and at an external pH of 8.0, where deltapH is inverted and little benzoate is taken up. Radiolabeled proteins were separated on 2-D gels and were identified on the basis of the index of VanBogelen and Neidhardt. In the absence of benzoic acid, little difference was seen between pH 6.5 and pH 8.0; this confirms that the mechanisms of protein homeostasis in this range are constitutive, including the transition between positive and inverted deltapH. Addition of benzoate at pH 6.5 increased the expression of 33 proteins. Twelve of the benzoate-induced proteins were induced at pH 8.0 as well, and nine of these matched proteins induced by the uncoupler dinitrophenol. Eighteen proteins were induced by benzoate only at pH 6.5, not at pH 8.0, and were not induced by dinitrophenol. One may be the iron and pH regulator Fur, which regulates acid tolerance in Salmonella spp. The other 13 proteins had not been identified previously. The proteins induced by benzoate only at a low pH may reflect responses to internal acidification or to accumulation of benzoate.     

Rapid access to biomedical knowledge with GeneCards and HotMolecBase: implications for the electrophoretic analysis of large sets of gene products

Rapid access to well-organized information about gene products is important for many studies that simultaneously monitor large sets of those factors, for example with electrophoretic methods. HotMolecBase and GeneCards, Internet resources that may be accessed from our Bioinformatics homepage at http://bioinfo.weizmann.ac.il/, have been designed to address similar problems. GeneCards presents semi-automatically collected information about all approved human genes and their products (with a focus on cellular functions and medical aspects), and offers a new kind of knowledge navigation guidance system that interactively guides the information-seeking scientist to relevant information. On the other hand, HotMolecBase is a collection of more extensive hypertext fact sheets about a small set of medically interesting molecules (mainly proteins) that are regarded as especially promising targets for drug development. Together, both resources may help scientists world-wide to find their way in the growing labyrinth of biomedical information on the World Wide Web. In the present article, we want to explain how these resources may be used by researchers who want to access information related to particular spots on two-dimensional electrophoresis gels.     

First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis

Data on the identification of proteins of Bacillus subtilis on two-dimensional (2-D) gels as well as their regulation are summarized and the identification of 56 protein spots is included. The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt- and ethanol stress as well as oxidative stress was analyzed. N-terminal sequencing of protein spots allowed the identification of 93 proteins on 2-D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions. A computer-aided analysis of the 2-D gels was used to monitor the synthesis profile of more than 130 protein spots. Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entirely different stimuli. The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B. subtilis. These investigations demonstrated that the alternative sigma factor sigma B is involved in the regulation of almost all of the general stress proteins and that the phoPR two-component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation.     

Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination

With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two-dimensional (2-D) gels corresponding to the most abundant yeast proteins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2-D gels. Some novel proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak-table data and peptide match-list data into databases enables the identification of a larger percentage of the peaks.     

From genome to proteome: protein map of Haemophilus influenzae

High-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis allows the separation of complex biological mixtures (i.e., several hundred proteins from a bacterial cell lysate) in a single experiment. In this report proteins from Haemophilus influenzae were separated by 2-D gels and analyzed by peptide mass fingerprinting and/or amino acid analysis. By comparing the peptide mass profiles and the amino acid composition with the Haemophilus influenzae database, 119 protein spots were identified. The combination of amino acid analysis and peptide mass fingerprinting is a powerful tool for a rapid and economical identification of a large number of proteins resolved by 2-D gels. Studies on gene regulation and changes of protein expression upon drug treatment require quick and serial analysis techniques to efficiently identify potential new drug targets.     

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.     

Fractionation on the microscale of brain polyribosomes by electrophoresis on acrylamide gels

A method is described for brain polyribosome fractionation by acrylamide gel electrophoresis. Brain polyribosomes were run in 2.0% gels in quartz capillaries of 800 mum inner diameter where the gels were supported by capillary force. The gels could then be ultraviolet-scanned in situ. Amounts of brain polyribosomes as small as 10-10(-3) A260nm unit could be analysed by this method. The method was checked by running a macroscale-prepared brain polyribosome sample. The various electrophoretic bands obtained showed a favourable A260nm: A280 ratio. A short RNase treatment caused the disappearance of the slowly migrating bands and the emergence of a predominant band migrating faster than the dimer. The various polyribosomal bands were then identified by comparison with the mobility of polyribosome fractions taken from a sucrose gradient fractionation. Finally, the electrophoretic pattern of brain polyribosomes compared favourably with the pattern obtained by the classic method of sucrose gradient sedimentation. The electrophoretic fractionation of polyribosomes prepared from one rat hippocampus (80 mg) is presented.     

SDS agarose gels for analysis of proteins

A new agarose-based protein electrophoresis gel system is described. The system consists of a highly resolving agarose, MetaPhor XR (FMC BioProducts, Rockland, ME, USA) dissolved in urea and TBE buffer and a stacking gel composed of a high gel-strength agarose, SeaKem Gold (FMC BioProducts). TBE containing sodium dodecyl sulfate (SDS) is used as electrophoresis buffer. The disadvantages of traditional agarose gels have been overcome, and several advantages over polyacrylamide gels have been demonstrated. The system is capable of high-resolution separation of small proteins and has a dynamic separation range equivalent to a 4%-20% gradient polyacrylamide gel. Furthermore, the staining of protein bands by Coomassie Brilliant Blue is very uniform in this gel, and depending on the protein, higher detection sensitivity can be obtained compared to SDS polyacrylamide gels. In Western blotting, proteins are more efficiently transferred to the membrane from the agarose gel than from polyacrylamide gels. Finally, the exceptional stability of agarose allows for gels to be precast and stored for a year.     

Membrane transport in rat liver endocytic pathways: preparation, biochemical properties and functional roles of hepatic endosomes

The endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol-treated rats, were analyzed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One- and two-dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the hepatocyte.     

A two-dimensional protein map of human amniotic fluid at 17 weeks' gestation

Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: IPG/SDS-PAGE) we provide a two-dimensional (2-D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver-stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques. This report details our investigation on AF proteins focusing on the 17th week of gestation, when AF is most commonly used for clinical evaluation of fetal disorders. As a whole, the map displays a number of potential markers for fetal development and for gestation abnormalities. The 2-D electrophoretic technique allows the monitoring of all these proteins at the same time along with additional spots that may prove of diagnostic significance.     

Proteins and their amino acid compositions: uniqueness, variability, and applications

Amino acid compositions (AAC) of proteins were analyzed in terms of their uniqueness and variability. Using several measures of convergence between the AACs of randomly chosen proteins versus those stored in protein data banks, it was established that certain families of proteins have unique AACs despite the mutations of their sequences which were imposed in the process of evolution. AACs may be used to establish the identities of many proteins which were sorted through various chromatographic media prior to their fractionation on two-dimensional (2D) gels. Subfractionations of proteins markedly enhance the chances for proper identification of low-abundant proteins which rest inaccessible if the total protein extract of an organ is analyzed on 2D gels. Although the amino acid composition versus protein identity (AAC-PI) method allows identification with high confidence of unique proteins resolved on monodimensional SDS-PAGE (1D) gels and arrays of protein isoforms resolved on two-dimensional (2D) gels, selective immunoblotting is still a more robust method. Thus, in principle, the AAC-PI method may allow limiting the number of "unknown" spots on 2D gels which could be further investigated by microsequencing and/or mass spectroscopy. However, to resolve certain ambiguities inherently linked with protein identities derived only from their AACs, the AAC-PI method must be sometimes aided by microsequencing and immunoblotting, especially in the construction of high-resolution 2D maps of proteins. A suite of algorithms which form the AAC-PI method are described in detail.     

Sequence analysis of betaA3, betaB3, and betaA4 crystallins completes the identification of the major proteins in young human lens

A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as alphaA, alphaB, betaA1, betaA3, betaA4, betaB1, betaB2, betaB3, gammaS, gammaC, and gammaD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human betaA1, betaA3, betaA4, and betaB3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of betaA3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.     

An alternative for purification of low soluble recombinant hepatitis C virus core protein: preparative two-dimensional electrophoresis

For isolation of low soluble recombinant full-length (amino acids 1-191) core protein of hepatitis C virus (HCV) overexpressed in Escherichia coli, the advantage of combining two electrophoretic techniques, in comparison with chromatographic separation, is demonstrated. The protein extract was first solubilized in agents compatible with electrophoretic separation. Using preparative liquid phase isoelectric focusing (IEF) the protein of interest was first concentrated within a defined acidic pH range. These fractions were then submitted to preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) to isolate the 22 kDa protein. The second-dimensional step allowed the isolation of 2 mg of the purified recombinant HCV core protein (rHCV-C191) from 1.5 g bacterial pellet. This quantity is sufficient to characterize the protein and to perform immunogenicity studies. This procedure of two-dimensional preparative electrophoresis is applicable to a wide range of biological samples and represents an alternative for purification of insoluble proteins.