Changes in cell surface proteins during differentiation of mouse embryonal carcinoma cells

The cell surface proteins of teratocarcinoma-derived embryonal carcinoma cells (ECC), of parietal endoderm (Pys-2 and F9-AC cl 9), and of fibroblasts (OTT6050f) were radioiodinated by a lactoperoxidase method and analyzed by two-dimensional gel electrophoresis. The combined electrophoretic profiles of proteins from a number of ECC lines allowed the determination of eight ECC-unique polypeptides. Parietal endoderm and fibroblast expressed their own unique polypeptides. The two parietal endoderm-specific polypeptides are identical to the subunits of laminin. Retinoic acid-induced differentiation of one ECC line (F9) resulted in the disappearance of polypeptides specific for ECC and the appearance of those specific for the parietal endoderm.     

Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers

Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases.     

Peptide mapping of heterogeneous protein samples.

A simple two-dimensional electrophoretic method for peptide mapping of heterogeneous protein samples is presented. The reduced and denatured proteins of the mixture are separated in a first dimension by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After completion of the electrophoresis, the whole gel lane is equilibrated in stacking gel buffer and is transferred at right angles onto a second slab gel. A protease solution is overlayed on the gel lane and a partial proteolysis of the proteins to be analyzed is performed during the stacking phase of the second electrophoresis. The second electrophoresis resolves the characteristic pattern of peptides of each individual protein as a series of spots located below the original position of the undigested protein. The peptide maps of the following samples are presented as examples: protein P23 and P23* of bacteriophage T4, membranes of Dictyostelium discoideum, membranes of human erythrocytes, and 35S-labeled proteins of D. discoideum synthesized in vivo or in a cell-free wheat germ extract. In complex samples, up to 20 individual proteins can be analyzed at once and a protein comprising only 1% of the total sample generates a clearly identifiable peptide pattern. Good reproducibility of the patterns obtained allows the comparison of samples of different origins.     

Analysis of the major polypeptides of spectrin by tryptic digestion

The two major polypeptides of erythrocyte membrane spectrin have been isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The tryptic peptide maps of the two polypeptides have been prepared by thin-layer chromatography and electrophoresis. Radioactive peptides have been prepared by ¹⁴C-carboxymethylation and chloramine T-catalysed ¹²⁵I iodination. Maps of both sets of peptides demonstrate a marked similarity between the two parent polypeptides.     

Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels

A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.     

Peptide mapping of basic proteins by proteolysis in acetic acid/urea-minislab polyacrylamide gels

A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications.     

Assignment of genes to the human X chromosome by the two-dimensional electrophoretic analysis of total cell proteins from rodent-human somatic cell hybrids.

The technique of two-dimensional (2-D) gel electrophoresis was sued to identify five human X-linked gene products in crude cell extracts of mouse-human and Chinese hamster-human somatic cell hybrids. The human origin of these five polypeptides was demonstrated by their comigration with human fibroblast proteins and their failure to comigrate with polypeptides in extracts from the mouse or hamster parental cells. All five polypeptides were present in extracts of rodent-human hybrids that contained a human X chromosome, but were not found in extracts of cells that lacked a human X chromosome. Chromosome analysis of the hybrid clones revealed that the human X chromosome is both necessary and sufficient for the expression of the five polypeptides, designated pX-24, pX-27, pX-37, pX-40, and pX-56. pX-56 can be identified as the human X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) (E.C.1.1.1.49), while polypeptides pX-24, pX-27, pX-37 and pX-40 have molecular properties unlike those of known human X-linked gene products. pX-24 appears to be a membrane-bound protein that maps to the distal portion of the long arm of the human X chromosome, while pX-27, pX-37, and pX-40 are soluble proteins that map to the proximal long arm or to the short arm of the human X chromosome. 2-D gel electrophoretic analysis of extracts from somatic cell hybrids provides a general method for identifying polypeptides in crude cell extracts coded for by any specific chromosome and can be used to study primary gene products not previously amenable to genetic analysis.     

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.     

Isolation of yeast ribosomal proteins L3 and L2 for immunological studies

We report on a rapid method for the isolation and purification of the yeast ribosomal proteins L3 and L2 using a simple instrumentation. Preparative dodecyl sulfate polyacrylamide gel electrophoresis was applied to the separation of cytoplasmatic ribosomal proteins of the large subunit from the yeast Saccharomyces cerevisiae. The polypeptides were removed from gel slices by electrophoretic elution. Subsequent analytical electrophoresis showed groups of proteins in all but two fractions. The latter were further analysed by a two-dimensional gel electrophoresis system which disclosed the purity of two polypeptides. They were identified as L3 and L2. Their molecular masses were 51.5 and 44 kDa as estimated from the gels. A possible application to the isolation of other yeast ribosomal proteins is discussed. An antiserum against the polypeptide L3 was raised in a rabbit. Applying an enzyme-linked immunosorbent assay (ELISA) we were able to determine the relative antibody concentration. Its specificity was demonstrated by immunoblotting.     

Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides

The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.     

Detection of transiently phosphorylated membrane proteins by protein blotting through a nonionic detergent layer

A rapid approach for detecting tentative membrane proteins which are transiently phosphorylated/dephosphorylated is described. Cell fractionation is unnecessary, as are other manipulations of sample preparation during which artifactual modifications or sample loss might occur. The method is shown to be useful for the detection of such phosphorylation during cellular response to the binding of specific ligand. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed successively through gels of different sieving sizes. These "primary" gels were then subjected to "detergent blotting," a variation of electroblotting in which polyacrylamide gel containing the nonionic detergent Nonidet-P40 (secondary gel) was inserted between the primary gel and a Zeta-Probe membrane. Phosphorylated interleukin 2 receptors were selectively retained in the secondary gel. Upon stimulation of human platelets with thrombin, at least 11 polypeptides were found to be rapidly phosphorylated/dephosphorylated using the method. Among them, five phosphorylated polypeptides were trapped in the secondary gel, suggesting that they might be membrane proteins. This technique should be useful to rapidly screen transiently phosphorylated/dephosphorylated membrane proteins which might be involved in membrane transductional signaling.     

Structural relationships of the major glycoproteins from human alveolar proteinosis surfactant

Alveolar proteinosis is a disease characterized by accumulation of proteinaceous material in the alveolar space of the lung. Two major collagenase-sensitive polypeptides, alveolar proteinosis peptides of 34 kDa kilodaltons (APP-34) and of 62 kDa (APP-62), were isolated from bronchioalveolar lavage of patients with alveolar proteinosis. These proteins co-purified during fast-performance liquid chromatography (FPLC) chromatofocusing and were separated from each other by electroelution following SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of these proteins demonstrated that both shared antigenic sites with the normal human surfactant-associated protein of Mr 34,000 (SAP-34) using both polyclonal and monoclonal antibodies generated against SAP-34. Removal of asparagine-linked oligosaccharides from the 34 kDa and 62 kDa alveolar proteinosis proteins with endoglycosidase F resulted in polypeptides of 28 kDa from APP-34 and 56 kDa from APP-62. Amino acid analysis and tryptic peptide maps of the electroeluted APP-34 and APP-62 proteins were essentially identical and similar to that previously reported for human SAP-34, supporting the likely relationship of APP-34 and APP-62 as monomer and dimer of the normal SAP-34. APP-34 and APP-62 were both sensitive to bacterial collagenase, yielding collagenase-resistant fragments of 21 kDa, similar in migration and amino acid composition to the fragment generated by collagenase digestion of normal human SAP-34. High molecular weight aggregates of APP-34 and APP-62 were the result of sulfhydryl-dependent and non-sulfhydryl-dependent cross-linking. A domain in the C-terminal non-collagenous portion of the molecules which forms sulfhydryl-dependent oligomers was identified. The two major polypeptides accumulating in the airway of patients with alveolar proteinosis are monomeric (34 kDa) and dimeric (62 kDa) forms of the major surfactant-associated glycoprotein, SAP-34.     

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Electrophoretic map of boar sperm plasma membrane polypeptides and localization and fractionation of specific polypeptide subclasses

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.     

Turnover of cytokeratin polypeptides in mouse hepatocytes

The turnover of cytokeratin polypeptides A (equivalent to No. 8 of the human cytokeratin catalog) and D (equivalent to human cytokeratin No. 18) of mouse hepatocytes was studied by pulse-labeling of mouse liver proteins after intraperitoneal injection of L-[guanido-14C]arginine and [14C]sodium bicarbonate. At various times after injection cytoskeletal proteins were prepared and separated by SDS-polyacrylamide gel electrophoresis, and the specific radioactivities of polypeptides recovered from excised gel slices were determined. With L-[guanido-14C]arginine a rapid increase in the specific radioactivity of both cytokeratins was observed which reached a plateau between 12 and 24 h. With [14C]sodium bicarbonate maximal specific radioactivity was obtained at 6 h followed by a rapid decrease to half maximum values within the subsequent 6 h and then a slower decrease. Half-lives were determined from the decrease of specific radioactivities after pulse-labeling by least-squares plots and found to be 84 h (for cytokeratin component A) and 104 h (component D) for arginine labeling. Values obtained after bicarbonate labeling were similar (95 h for A and 98 h for D). These results show that liver cytokeratins are relatively stable proteins and suggest that components A and D are synthesized and degraded at similar rates, probably in a coordinate way.     

Proteomics based on peptide fractionation by SDS-free PAGE

Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.     

Correlated induction of nitrate uptake and membrane polypeptides in corn roots

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the ³⁵S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.     

Multiple forms of polyoma virus tumor antigens from infected and transformed cells.

At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.     

Characterization of the protein components of Nephila clavipes dragline silk

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.