A noncomputerized scanning method for determining relative protein quantities and synthesis rates on two-dimensional electrophoretic gels

A densitometric method that permits the determination of relative amounts and synthesis rates of proteins separated by two-dimensional gel electrophoresis is presented. The method is applicable to proteins that have been stained by Coomassie blue or proteins that have been radiolabeled. The analysis is achieved by scanning selected spots with an ordinary densitometer in the horizontal and vertical direction and relating the response to the volume of an ellipsoid. A linear dependence is observed between the amount of protein or incorporated radioactivity and the measured optical density. The advantage of this method is that specialized scanning instruments and computer analysis are not required. The method is most useful for the analysis of a few specific proteins which change in their relative amount or specific activity due to experimental manipulations. Difficulties in the analysis of protein spots derived from the twodimensional gel electrophoresis technique are discussed and compared to an analysis of bands from the one-dimensional electrophoresis technique.     

pH-dependent protein profiles of Helicobacter pylori analyzed by two-dimensional gels

Background. Helicobacter pylori survives transient exposure to extreme acid prior to adherence and growth on the gastric epithelium at neutral pH.
Materials and Methods. The effect of pH stress on protein profiles of H. pylori was observed using two-dimensional gel electrophoresis (2-D gels). H. pylori 26695 was grown microaerobically in tryptone-yeast extract broth, 3% fetal bovine serum. Growth in acid alkalinized the medium, whereas growth in base caused acidification. For 2-D gel analysis of protein profiles, cultures were grown in media buffered at pH 5.7 and at pH 7.5.
Results. Under all pH conditions, the most abundant proteins observed were the urease structural subunit UreB and the chaperonin GroEL. Growth in acid significantly increased the abundance of UreB. Thus, urease expression is not completely constitutive, as reported previously, but shows regulation by pH. Another protein observed only at low pH was identified as mammalian apolipoprotein A-I, possibly taken up by H. pylori from bovine serum in the growth medium. This finding, if confirmed, suggests that uptake of high-density lipoprotein from the human host may facilitate acquisition of cholesterol, required for formation of the unique cholesteryl glucosides in the membrane of H. pylori. In growth above pH 7, three stress proteins were induced: GroES (HspA), GroEL (HspB), and the antioxidant AhpC homolog TsaA. In addition, N-terminal sequence analysis identified five additional proteins that had not previously been reported on 2-D gels of H. pylori (FMN, SodB, TrxB, TsaA, and Tsr).
Conclusions. In summary, our 2-D gel study reveals expression of several proteins dependent on growth pH.     

Evaluation of individual protein errors in silver-stained two-dimensional gels

The relationship between spot volume and variation for all protein spots observed on large format 2D gels when utilising silver stain technology and a model system based on mammalian NSO cell extracts is reported. By running multiple gels we have shown that the reproducibility of data generated in this way is dependent on individual protein spot volumes, which in turn are directly correlated with the coefficient of variation. The coefficients of variation across all observed protein spots were highest for low abundant proteins which are the primary contributors to process error, and lowest for more abundant proteins. Using the relationship between spot volume and coefficient of variation we show it is necessary to calculate variation for individual protein spot volumes. The inherent limitations of silver staining therefore mean that errors in individual protein spot volumes must be considered when assessing significant changes in protein spot volume and not global error.     

JVirGel: Calculation of virtual two-dimensional protein gels

We developed JVirGel, a collection of tools for the simulation and analysis of proteomics data. The software creates and visualizes virtual two-dimensional (2D) protein gels based on the migration behaviour of proteins in dependence of their theoretical molecular weights in combination with their calculated isoelectric points. The utilization of all proteins of an organism of interest deduced from genes of the corresponding genome project in combination with the elimination of obvious membrane proteins permits the creation of an optimized calculated proteome map. The electrophoretic separation behaviour of single proteins is accessible interactively in a Java(TM) applet (small application in a web browser) by selecting a pI/MW range and an electrophoretic timescale of interest. The calculated pattern of protein spots helps to identify unknown proteins and to localize known proteins during experimental proteomics approaches. Differences between the experimentally observed and the calculated migration behaviour of certain proteins provide first indications for potential protein modification events. When possible, the protein spots are directly linked via a mouse click to the public databases SWISS-PROT and PRODORIC. Additionally, we provide tools for the serial calculation and visualization of specific protein properties like pH dependent charge curves and hydrophobicity profiles. These values are helpful for the rational establishment of protein purification procedures. The proteomics tools are available on the World Wide Web at http://prodoric.tu-bs.de/proteomics.php.     

Rapid identification of medically important yeasts by electrophoretic protein patterns

A simple electrophoretic method for yeast identification was evaluated. Whole cells were extracted by SDS and the protein profiles obtained in SDS-PAGE after Coomassie blue staining were compared for 52 strains from 9 species of yeast or yeast-like fungi commonly isolated from man (Candida albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. tropicalis, Geotrichum candidum, Saccharomyces cerevisiae). The corresponding patterns showed 30 to 45 polypeptides in the range 95-20 kDa and were clearly different for the 9 species. No differences could be detected between strains from the same species. The characteristic patterns were obtained within 24 h allowing rapid identification of the most commonly encountered clinical yeast isolates.     

Characterization of Spiroplasmas by one- and two-dimensional protein analysis on polyacrylamide slab gels

Differences betweenSpiroplasma citri isolates were detected by one-dimensional electrophoresis of proteins on gradient polyacrylamide slab gels. Two-dimensional protein maps (electrofocusing followed by electrophoresis) showed a highly characteristic pattern for allS. citri isolates examined. Coanalysis of mixed protein samples from pairs ofS. citri strains revealed more than 150 comigrating proteins common to allS. citri isolates, but also a number of noncomigrating proteins. Some noncomigrating proteins were present in one isolate but not in another, while other proteins whose migrational properties were only slightly different from one isolate to the other (homologous proteins), were present in more than one isolate.S. citri isolates had many common and only a few homologous proteins. Comparisons ofS. citri with the corn stunt spiroplasma revealed few common proteins and a large number of homologous proteins. When comparingS. citri and the suckling mouse cataract spiroplasma, few common and homologous proteins were apparent. However, several of these common proteins were also shared by the corn stunt spiroplasma, suggesting that they may well represent genus-specific proteins. The data also offer additional evidence that the suckling mouse cataract spiroplasma differs significantly fromS. citri and corn stunt spiroplasmas and probably deserves a separate species designation.     

Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: Strategies and applications

The entire genomic DNA sequences of a number of prokaryotic and eukaryotic species are now available and many more, including the human genome, will be completed in the near future. The state-of-life of a cell at any given time, however, is defined by its protein composition, i.e., its proteome. Gel electrophoresis, mass spectrometry, and bioinformatics will be important tools for protein and proteome analysis in the post-genome era. Protein identification from electrophoretic gels by mass spectrometric peptide mapping or peptide sequencing combined with sequence database searching is established and has been applied to numerous biological systems. We describe current strategies and selected applications in molecular and cell biology. The next challenges are detailed structure/function analyses, which include studying the molecular composition of multiprotein complexes and characterization of secondary modifications of proteins. The advantages and limitations of a number of mass spectrometry-based strategies designed for microcharacterization of low amounts of protein from electrophoretic gels are discussed and illustrated by examples. Proteins Suppl. 2:74–89, 1998. © 1998 Wiley-Liss, Inc.     

Correlation analysis of two-dimensional gel electrophoretic protein patterns and biological variables

Background  

Two-dimensional gel electrophoresis (2DE) is a powerful technique to examine post-translational modifications of complexly modulated proteins. Currently, spot detection is a necessary step to assess relations between spots and biological variables. This often proves time consuming and difficult when working with non-perfect gels. We developed an analysis technique to measure correlation between 2DE images and biological variables on a pixel by pixel basis. After image alignment and normalization, the biological parameters and pixel values are replaced by their specific rank. These rank adjusted images and parameters are then put into a standard linear Pearson correlation and further tested for significance and variance.     

Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

Fatty acid-binding protein from human heart localized in native and denaturing two-dimensional gels

Summary A group of low molecular weight fatty acid-binding cytosolic proteins, FABP_c with high abundance in heart, liver, skeletal muscle, intestine and adipose tissue, are anticipated to play a role in long-chain fatty acid metabolism in these tissues. Recently, a FABP_c with M_T 15 kDa has been purified from human heart muscle and found to be present in levels 2–4% of cytosolic proteins of human heart myocytes. In the present study two-dimensional gel electrophoresis under native and denaturing conditions has been used to characterize FABP_c from human heart and this protein is found to be a major protein of human heart myocytes. The pI of FABP_c from human heart was found to be about 5.3 under native conditions and about 6.5 in the presence of 9 M urea.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase

This study developed an enzymatic method for high-throughput mapping of phosphoproteins on two-dimensional (2-D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2-D electrophoresis. Phosphoproteins could be mapped on the 2-D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine-rich tetratricopeptide repeat-containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high-throughput mapping of phosphoproteins in proteome research.     

A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels

DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein-nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous (32)P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.     

A high-affinity protein stain for western blots, tissue prints, and electrophoretic gels.

A method for protein staining using copper phthalocyanine 3,4',4',4'-tetrasulfonic acid tetrasodium salt is described. The procedure is applicable to protein blots and tissue prints, as well as to polyacrylamide and agarose gels. It is also simple, involving only application of the stain and rinsing. For protein blots and tissue prints the staining is rapid, taking less than 1 min to completion, and more sensitive than any previously described dye-based nonspecific protein staining system. The staining is easily reversible, requiring only a change in pH to remove the dye.     

Double-labeling and high precision comparison of complex protein patterns on two-dimensional polyacrylamide gels

Combination of double-labeling and two-dimensional polyacrylamide gel electrophoresis allows high precision comparison of proteins from two different cultured cell lines. Extracts of cell lines labeled with ¹⁴C and ³H, respectively, are mixed and analyzed on a single two-dimensional gel. Fluorography detects both isotopes, but autoradiography through carbon paper detects only ¹⁴C. Proteins can be classified into classes common to both cell lines and those specific to each cell line. The method should be useful for a wide range of biochemical studies.     

Serial electrophoretic transfers: A technique for the identification of numerous enzymes from single polyacrylamide gels

We describe an electrophoretic method for the transfer of macromolecules from polyacrylamide slab gels to ion-exchange paper without loss of clarity or resolution. A series of partial transfers provides numerous copies of a single gel separation, and these copies may be assayed independently with enzyme specific stains. This method therefore combines the advantages of polyacrylamide gel electrophoresis for detecting enzyme variation with an efficient method for examining numerous enzymes from a single gel separation.This work was supported by NIH Grants GM 24849 and GM 27119 to R. C. Lewontin.     

Interaction of TMV-proteins during electrophoretic separation in polyacrylamide gels

Summary The technique of polyacrylamide gel electrophoresis was applied to demonstrate interaction between proteins of different strains of tobacco mosaic virus. The advantages and limitations of the method for such studies have been indicated. The results are discussed in connection with the role of primary structure of TMV-proteins for mixed aggregations.The present observations were reported by us under the title, Mixed Aggregation of Protein Sub-Units of Tobacco Mosaic Virus Mutants: Role of Primary Structure, during a symposium on Interaction between Subunits of Biological Macromolecules, organized by the International Union of Pure and Applied Biophysics, and held in Cambridge, U.K., from 24–27th June, 1968.