Quantitative determination of overlapped proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A method for resolving an overlapped polypeptide pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis was described. The procedure was essentially a Gaussian fitting using the least squares method, and could resolve more than 20 overlapped components simultaneously. The applicability to overlapped and shouldered patterns was evaluated using practical electrophoretic data with varying amounts of mitochondrial samples. The relative contents of respective polypeptide components gave a good agreement regardless of the loaded amounts.     

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 M urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.     

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.     

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.     

Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Efficient protein folding and quality control are essential for unperturbed cell viability. Defects in these processes may lead to production of aberrant polypeptides that are either degraded leading to “loss-of-function” phenotypes, or deposited in or outside cells leading to “gain-of-toxic-function” phenotypes. Elucidation of molecular mechanisms regulating folding and quality control of newly synthesized polypeptides is therefore of greatest interest. Here we describe protocols for metabolic labelling of transfected/infected mammalian cells with [³⁵S]-methionine and [³⁵S]-cysteine, for immunoisolation from detergent extracts of the selected model proteins and for the investigation of the model polypeptide’s intracellular fate in response to chaperone-deletions or to cell exposure to folding or degradation inhibitors.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Purification of NADPH-specific indole-3-acetaldehyde reductase from Cucumis sativus by two-dimensional native polyacrylamide gel electrophoresis

NADPH-specific indole-3-acetaldehyde (IAAId) reductase from cucumber ( Cucumis sativus L. 相似文献   

Sequence homology analysis of proteins by chemical cleavages: using a mono and two dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The examination of possible sequence homology in proteins using SDS-PAGE systems after chemical cleavage is described. After SDS-PAGE, the establishment of amino acid compositions, the techniques of staining gel and five different methods of chemical cleavages (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid and nitrothiocyano benzoic acid) have been used for peptide mapping studies. Potential applications of this technique are discussed from both the biochemical and immunochemical point of view.     

A rapid capillary gel electrophoresis method for the quantitative determination of RuBisCo in spinach

A capillary gel electrophoretic (CGE) method for the quantitative analysis of RuBisCo in spinach leaves was developed. RuBisCo was resolved into large and small subunits in the presence of sodium dodecyl sulphate (SDS) by the CGE procedure which enabled the determination of the molecular weight of each unit accurately; the values so determined were in close agreement with those reported using other methods. Advantages of CGE over SDS-polyacrylamide gel electrophoresis and high-pressure gel filtration include decreased sample preparation and analysis time, superior resolution and greater sensitivity permitting reduced sample size and trace analysis. In addition, CGE provided precise quantification of RuBisCo and was demonstrated to be a viable alternative to other available methods of protein analysis.     

Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS; the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539–1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ. Received: 25 July 1998 / Accepted: 22 September 1998     

Comparative analysis of amplified fragment length polymorphism and pulsed field gel electrophoresis in a hospital outbreak and subsequent endemicity of ampicillin-resistant Enterococcus faecium

Reliable molecular methods for determination of relatedness between bacterial isolates have become increasingly important to evaluate outbreaks and endemic situations with nosocomial pathogens. In the present study Simpson's index of diversity with calculated confidence intervals was used to compare amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) analysis of a hospital outbreak of ampicillin-resistant Enterococcus faecium and subsequent endemicity. The outbreak, in a Norwegian tertiary hospital, of infections caused by these enterococci started in 1995 and increased in 1996 after which the situation turned endemic. The purpose of this study was to compare the two methods in this setting and to determine the length of time during an outbreak that these methods are sufficiently valid to be of value for hospital infection control efforts. One hundred and sixty clinical isolates from urine specimens collected during the period 1995-1999 were included. The findings indicate that PFGE and AFLP are equally discriminative and could in this setting be used for typing purposes over the whole 5-year period.     

Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system

A procedure for examining possible sequence homology between two or more proteins in a heterogenous protein mixture using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Three different chemical reagents (cyanogen bromide, hydroxylamine, and acetic acid) and three enzymes (α-chymotrypsin, trypsin, and Staphylococcus aureus protease) have been used as the cleavage reagents for the peptide mapping studies. Potential application of this technique in conjunction with radioactive labeling and immunological studies was also demonstrated.     

A comparative investigation of soluble protein polymorphism in Robinia pseudoacacia roots by polyacrylamide gel electrophoresis

Investigations of soluble proteins by polyacrylamide gel electrophoresis of root extracts of black locust (Robinia pseudoacacia L.) were carried out with 41 trees from diverse habitats representing dominant-stem forms (R. p. var. rectissima Raber) and typical forms (R. pseudoacacia L.). Soluble protein patterns of dominantstem forms and typical trees did not show differences attributable to tree form. Heritability estimates (broad sense) were determined as 9·19% within location and 7·.5% among populations. A variance components model was constructed which showed the interaction between parental trees and location to be most significant in determining variation. Location variance was second in importance, with parental variance and experimental error of less significance. The data were analyzed by the moment of inertia. It is indicated that, based on protein similarity, the dominant-stem form is an ecological variant and should not be given varietal status.     

A sodium dodecyl sulfate--polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000

A discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described which provides superior resolution of polypeptides with molecular weights from approximately 2500 to 90,000. The system utilizes a relatively low-mobility acetate ion in the stacking gel and high-mobility strong anions, sulfate and chloride, as leading and trailing ions in the separating gel. The entire system is run at pH 7.8. The separating gel contains 8 M urea, and can be used at acrylamide concentrations from 5 to 18%, all with 5% crosslinker concentrations. Using a number of protein standards, the calibration curves obtained with this system are linear over the molecular weight range from 2500 to 90,000, regardless of acrylamide concentration. These studies indicate that by providing good resolution of small peptides, this system greatly extends the utility of one-dimensional peptide mapping techniques.     

桔小实蝇幼虫总蛋白双向电泳技术体系的建立和优化

【目的】建立和优化桔小实蝇幼虫Bactrocera dorsalis(Hendel)总蛋白的双向电泳条件。【方法】使用BPP法和3种TCA-丙酮法(TCA-丙酮-A法:直接加入裂解液磨样;TCA-丙酮-B法:样品提取液中加入40 mmol/L DTT;TCA-丙酮-C法:样品提取液中加入0.07%β-巯基乙醇)提取桔小实蝇幼虫总蛋白;使用13 cm和24 cm p H 4~7的IPG胶条分离桔小实蝇幼虫总蛋白;使用考马斯亮蓝法及硝酸银染色法对双向电泳凝胶进行染色;使用5800 MALDI-TOF-TOF MS/MS质谱分析仪对BPP法获得的特异蛋白进行质谱鉴定,并将检索数据库物种分别设为Metazoa(Animals)与Drosophila(Fruit flies)进行数据库检索。【结果】TCA-丙酮法中,TCA-丙酮-C法提取效果最好,BPP法优于所有TCA-丙酮法;使用考马斯亮蓝染色与硝酸银染色效果相当;使用24 cm胶条的蛋白分辨率明显高于13 cm胶条;检索数据库物种设为Metazoa(Animals)可获得比Drosophila(Fruit flies)更加全面的信息。【结论】使用24 cm p H 4~7的IPG胶条对BPP法提取的桔小实蝇幼虫总蛋白进行双向电泳,采用考马斯亮蓝法对双向电泳凝胶进行染色,可得到更好的双向电泳图谱,检索数据库时检索物种可优先设为Metazoa(Animals)。     

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

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

Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis

The solubilizing power of various nonionic and zwitterionic detergents as membrane protein solubilizers for two-dimensional electrophoresis was investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf membrane proteins were used as model systems. Efficient detergents could be found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar heads. Among the commercially available nonionic detergents, dodecyl maltoside and decaethylene glycol mono hexadecyl ether proved most efficient. They complement the more classical sulfobetaine detergents to widen the scope of useful detergents for the solubilization of membrane proteins in proteomics.     

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

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