High-resolution two-dimensional electrophoretic analysis of acidic,basic and surface proteins of mouse neutrophils

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 200 proteins detected) and with fluorography or autoradiography after [³⁵S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of ¹²⁵I associated with actin) yielded more than 20 major ¹²⁵I-labelled proteins on high-resolution electrophoretic maps. The major ¹²⁵I-labelled protein (M_r ≈ 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [³⁵S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.     

High resolution two-dimensional electrophoresis of basic as well as acidic proteins.

A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.     

Protein species of the parvovirus minute virus of mice strain MVMp: involvement of phosphorylated VP-2 subtypes in viral morphogenesis.

The pattern of induced protein species of the prototype strain of the parvovirus minute virus of mice was determined in permissive A9 mouse fibroblast cells by high-resolution two-dimensional gel electrophoresis. Identities of the viral proteins in the gels were assigned by probing two-dimensional blots with antisera raised against either purified capsids (recognizing VP-1 and VP-2) or specific coding regions of the nonstructural proteins (NS-1 and NS-2) expressed as beta-galactosidase fusion products in bacteria. All viral proteins showed posttranslational modifications, phosphate being a common substituent. The NS-1 protein migrated as a basic polypeptide in the pI range of 7.4 to 7.8 with multiple stages of modification and as a likely minor but hyperphosphorylated component in the neutral region of the gel. The NS-2 isoforms were resolved at a pI value close to 5.5 as three groups of unevenly phosphorylated polypeptides, each composed of at least two protein species. Both VP-1 and VP-2 structural polypeptides were induced as heterogeneous phosphoproteins. The major VP-2 protein could be resolved in the form of a consistent pattern of three abundant (a to c), two intermediate (d and e), and one meager (f) neutral isoelectric focusing species or subtypes. This posttranslational modification precedes and is uncoupled from viral assembly, and all of the VP-2 subtypes are packaged into empty capsids at the induced stoichiometry. However, intracellular full virions harbored additional phosphorylated subtypes (g to l) and a subtle rearrangement in the whole VP-2 composition, while mature virions purified from lysed cultures lacked these subtypes, coordinately with the emergence of six neutral VP-3 subtypes. Thus, the virion coat undergoes a chemical transition entailed by genome encapsidation, in which phosphates seem to play a major role, triggering the preferential proteolytic cleavage of the more acidic VP-2 subtypes to VP-3. Parvoviruses, with small coding capacity, may regulate some morphogenetic steps, such as assembly, genome encapsidation, and maturation, by posttranslational modifications of their structural proteins.     

About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis

The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels.     

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.     

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve,brain, and spinal cord during development

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.     

Some improvements in two-dimensional gel electrophoresis of proteins. Protein mapping of eukaryotic tissue extracts.

In the course of adapting O'Farrell's (1975, J. Biol. Chem.250, 4007–4021) two-dimensional separation technique for proteins to eukaryotic material, we have made some modifications. During sample preparation, sodium dodecyl sulfate (SDS) can be included, with a resulting enhancement in reproducibility of gel patterns. However, heating in the presence of SDS leads to artifactual spots in the gels, probably as a result of protein charge modifications. Ultracentrifugation reduces the clogging at the top of the isoelectric focussing gel. For electrophoresis, some modifications of apparatus and technique are suggested. For the analysis of gels, a simple high-efficiency method for the counting of radioactivity in spots from dried gel slabs is described. In addition, an inexpensive microdensitometer option is described for the analysis of the autoradiographs. Patterns of proteins obtained from superior cervical sympathetic ganglia of rats and from other eukaryotic tissues are illustrated. Finally, a few of the proteins commonly found in mammalian tissue are identified on the gels.     

Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.     

Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry

A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.     

Microheterogeneity of Micro tubule-Associated τ Proteins Is Due to Differences in Phosphorylation

We have studied the heterogeneity of the microtubule-associated tau proteins using tau-specific antibodies and two-dimensional electrophoresis. Both monoclonal and polyclonal antibodies to tau proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sulfate (SDS)-polyacrylamide gels, with apparent molecular weights between 56,000 and 66,000. Immunoblots of cow brain microtubules separated on two-dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-gel electrophoresis in the second, reveal that greater than 30 isoforms of tau exist. The tau proteins vary in pI from 6.5 to 8.5, with the higher-molecular-weight forms being more acidic. The microheterogeneity of tau is not induced by cycling of microtubules, because two-dimensional immunoblots of tau from total brain are almost identical to those of tau from cycled tubules. Adult rat brain tau, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does tau from 7-day-old rats, which appears as only one band on SDS gels. After dephosphorylation of cow brain tau with alkaline phosphatase, the highest-molecular-weight band disappears on SDS gels. On two-dimensional gels, the number of tau variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with tau labeled in vivo with 32PO4 also indicate that the more acidic tau proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of tau proteins exists at all ages and is due, at least in part to differences in the state of phosphorylation of tau isoforms.     

The determination of similarities in amino acid composition among proteins separated by two-dimensional gel electrophoresis

A simple and rapid procedure has been developed to determine similarities in amino acid composition among cellular proteins separated by two-dimensional gel electrophoresis. Cells in tissue culture are simultaneously labeled with two different amino acids each tagged with a different radioisotope. The proteins are then separated on two-dimensional gels and their location on the gels determined by Coomassie-blue staining or autoradiography. Elution of the protein from the appropriate region of the gel followed by liquid scintillation counting yields an isotope ratio which reflects the ratio of the two amino acids in the protein. Examples of the use of this technique in analyzing mutant proteins, proteins altered by carbamylation, and cell proteins with similar amino acid composition (e.g., actin and tubulin) are given.     

Ultra-high sensitivity multi-photon detection imaging in proteomics analyses

We report on the use of 125I and 131I labeling and of new, multicolor, multi-photon detection (MPD) methods to routinely and quantitatively detect protein spots on two-dimensional gel electrophoresis plates in the zeptomole to attomole range. We demonstrate that the MPD methodology can be used to detect radioactive labels on two-dimensional gels and has several characteristics that are advantageous for functional proteomics. First, by using single particle detectors, the sensitivity for detection of radiolabels can be improved dramatically. Second, because single particle detectors can differentiate the particle energies produced by different decay processes, it is possible to choose combinations of radioisotopes that can be detected and quantified individually on the same 2-D gel. Third, the MPD technology is essentially linear over six to seven orders of magnitude, i.e., it is possible to accurately quantify radiolabeled proteins over a range from at least 60 zeptomoles to 60 femtomoles. Finally for radionuclides that decay by electron capture, e.g., with emission of both beta and gamma rays, co-incident detection of two particles/photons can be used to detect such radionuclides well below background radiation levels. These methods are used to monitor acidic/phosphorylated proteins in as little as 60 ng of HeLa cells proteins.     

Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.     

Two-dimensional maps in very acidic immobilized pH gradients

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185–192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8–5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimensions gels and for a proper silver staining of 2-D maps with practically no background deposition.     

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Fractionation of liver proteins by preparative electrophoresis

Summary. Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.     

Structural alterations of acidic proteins by acid treatment of chromatin

Summary Recent studies into the properties and biological function of the acidic (non-histone) chromatin proteins have utilized inorganic or organic acids to first remove the histones prior to analysis of the acidic proteins. Examination of the effects of the acid treatment on the DNA and acidic proteins by immunochemistry, circular dichroism, and the ability of the DNA to serve as a template in thein vitro DNA-dependent RNA synthesis, has demonstrated a marked structural change (denaturation) in the proteins and DNA after the acid treatment. Other methods of removing histones, e.g., by high salt or salt and urea, are recommended for studies, especially for those of the biological functions, of the DNA and acidic proteins.