Quantitative predictions for DNA two-dimensional display according to size and nucleotide sequence composition

2-D DNA display is a simple separation method that provides a fast and economical way of visualizing polymorphism and comparing genomes. The DNA fragments are separated first according to their size by standard gel electrophoresis and then according to their sequence composition using denaturing gradient gel electrophoresis. First developed by Fischer and Lerman (Cell 1979, 16, 191-200), this method has recently been used to distinguish strains within a bacterial species. The genomic restriction fragments are displayed as spots on a 2-D surface. Although most of the relevant physical mechanisms are understood, this technique is mostly empirical and remains essentially qualitative. In view of optimizing this procedure, we combine our understanding of the different physical mechanisms at play to develop a complete numerical model to predict the relative coordinates of the spots as a function of the corresponding DNA sequence and of the experimental conditions. We experimentally validate our model by predicting the outcome of a 2-D display of the lambda phage genome. It thus becomes possible to optimize in silico the experimental parameters, to predict whether specific mutations as well as yet undescribed genetic polymorphisms can be resolved, and to assist in interpreting the experimental data.     

Determination of protein spots separated by two-dimensional polyacrylamide gel electrophoresis

A simple method for quantitating proteins in the spots on two-dimensional polyacrylamide gel electropherograms is described. The system consists in three steps: (1) O'Farrell's two-dimensional gel electrophoresis of the proteins to be analysed; (2) staining of the gels with Coomassie brilliant blue; and (3) determination of the area and integrated density of the stained spots by the Joyce Loebl Magiscan-1 image analysis system. The method can be used for the determination of proteins in the range 0.5-100 micrograms/cm2; the amount of protein involved in most spots detected by the staining method actually falls within this range. As the minimum spot diameter that can easily be handled by the method is about 2 mm, as much as 30 ng of protein in such a spot can be determined. The method can also be applied to autoradiograms.     

Degradation of individual intracellular proteins analyzed by two-dimensional gel electrophoresis and computerized video densitometry

A technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse-chase-labeling of cells using radioactive amino acids [35S]methionine, two-dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two-dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell-type-specific proteins were significantly enhanced (p less than 0.01) in fibroblast cultures of Duchenne muscular dystrophy origin.     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

Rational approach to quantitative sodium dodecyl sulfate capillary gel electrophoresis of monoclonal antibodies

Sodium dodecyl sulfate capillary gel electrophoresis has been used to separate and quantify murine monoclonal antibodies. The method uses a murine IgG, whose subclass differs from the analyte antibody, as an internal reference. The internal reference is chosen based on knowing that mouse IgG1 can be separated from mouse IgG2a or IgG2b. Good intra- and inter-day reproducibility [relative standard deviation (RSD)<2%] of peak-area ratio has been obtained. A calibration curve also demonstrates high linearity (R2=0.9999) of response for the analyte. The described method is highly suitable for accurate determination of the antibody concentration even if a capillary electrophoresis apparatus is unable to provide good injection reproducibility.     

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories.     

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.     

Polymer solution-filled column for the analysis of antisense phosphorothioates by capillary electrophoresis

A capillary electrophoresis (CE) column filled with 13% poly(ethylene glycol) (PEG) solution is demonstrated to resolve different lengths of antisense phosphorothioates in 100 mM Tris-borate (pH 9.0) buffer containing 30% formamide at 50 degrees C. Two sets of mixtures composed of 15-20 mers of either antisense phosphorothioate or phosphodiester oligonucleotides were synthesized based on a sequence of the antisense orientation directed against DNA-methyltransferase (denoted as MT-AS) and were used as model compounds. It was found that column coating reduced electroosmotic flow, as well as wall adsorption, and led to the separation of both phosphorothioate and phosphodiester molecules. Substantial peak broadening, however, specifically occurred to the phosphorothioates and was reduced statisfactorily by the addition of formamide into the buffer solution, raising the temperature, and raising the pH value. Under experimental conditions, a linear relationship between the migration time and the base number was observed, indicating that no peak compression artifacts existed. Without tedious pretreatment, antisense phosphorothioates were spiked into human serum, followed by water dilution, and then directly injected into the column. Separation of different lengths of phosphorothioates was observed using pressure injection, which did not suffer from injection bias.     

DNA sequencing

Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.     

A statistical model for unwarping of 1-D electrophoresis gels

A statistical model is proposed which relates density profiles in 1-D electrophoresis gels, such as those produced by pulsed-field gel electrophoresis (PFGE), to databases of profiles of known genotypes. The warp in each gel lane is described by a trend that is linear in its parameters plus a first-order autoregressive process, and density differences are modelled by a mixture of two normal distributions. Maximum likelihood estimates are computed efficiently by a recursive algorithm that alternates between dynamic time warping to align individual lanes and generalised-least-squares regression to ensure that the warp is smooth between lanes. The method, illustrated using PFGE of Escherichia coli O157 strains, automatically unwarps and classifies gel lanes, and facilitates manual identification of new genotypes.     

用凝胶色谱法测定聚N-乙烯咔唑中残留单体含量

本文用 GPC方法测定了聚 N-乙烯咔唑中残留单体含量,测定精确度约为±2％,聚合物中单体的最小检知量为0.03％。并且讨论了凝胶的孔径、溶剂纯度及不同检测器对结果的影响。     

A simple method for determination of the concentration of anti-dextran IgG in antiserum by means of affinity electrophoresis

A simple technique for determination of the concentration of anti-dextran IgG in antiserum using affinity electrophoresis is described. In the presence of an excess of intermediary molecular size dextran in the polyacrylamide gel, polyclonal anti-dextran IgG migrated in a single sharp band, separated from the nonspecific IgG fraction and other serum protein fractions. With this technique, 1-10 micrograms anti-dextran IgG in antisera can be determined within 3 h. We call the procedure ligand saturating affinity electrophoresis.     

Improved method for identification of proteins using two-dimensional electrophoresis with immobilized pH gradient isoelectric focusing.

Recent advances in protein sequence analysis now permit the determination of partial N-terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two-dimensional electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.     

Localization of separated protein bands in unstained electrophoresed polyacrylamide gradient slab gel.

Polyacrylamide gel electrophoresis has become the most widely used separation method in biological science. Once electrophoresis is complete the protein bands must be localized prior to excision. A zig-zag gel cutter is described which cuts a strip of gel from the side of a slab gradient gel or a gel of uniform concentration in peaks and valleys. The location of the protein of interest is determined by counting the number of peaks on the stained side strip. The portion of the unstained gel corresponding to the same count (number of valleys) is cut to recover the protein of interest from the main gel for further manipulations.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell – the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated. Received: 16 December 1999 / Accepted: 17 December 1999     

Development of a chip-based capillary gel electrophoresis method for quantification of a half-antibody in immunoglobulin G4 samples

A method based on microfluidic technology was developed to support quantitative analysis of recombinant monoclonal immunoglobulin G4 (IgG4) antibody samples. The assay was performed on an Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip Kit and the Protein 200 Plus assay software. Capillary electrophoresis principles have been transferred to a chip format that integrates all separation, staining, virtual destaining, and detection steps. The method is referred to in this paper as chip-based capillary gel electrophoresis (GelChip-CE method). The GelChip-CE method under nonreducing conditions proved to be a quantitative test for half-antibody determination in IgG4 samples. Similar to the traditional nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, the GelChip-CE method includes a denaturing step prior to separation. We showed that denaturing the sample by heating resulted in an artificial increase in the amount of half-antibody detected, which could be prevented by addition of N-ethylmaleimide to the sample buffer. The GelChip-CE method allowed for analysis of IgG4 samples with more accuracy, higher precision, and a faster turnaround time than SDS-PAGE and reversed-phase high-performance liquid chromatography (RP-HPLC).     

Two-dimensional electrophoresis in small gels for applications in veterinary medicine.

A versatile method for two-dimensional electrophoresis that can be performed easily, even in small routine laboratories, is described. The procedure combines a first-dimensional isoelectric focusing run in a PhastSystem with denaturing electrophoresis in a small vertical electrophoresis chamber. The described arrangement of two first-dimensional gel strips in the second dimension allows direct comparison of two related samples, eliminating most of the artifacts that usually lead to misinterpretations. The presented procedure can be conveniently adapted to the needs of each laboratory; at present, it is being extensively used to establish protein patterns of healthy individuals of different species, a prerequisite with regard to help and support diagnosis.     

Sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization-quadrupole time of flight-tandem mass spectrometry

Oligosaccharides were derivatized by reductive amination using benzylamine and analyzed by nanoelectrospray ionization-quadrupole time of flight-tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+H]+ ions for all benzylamine-derivatized oligosaccharides. To obtain structural information from these derivatized oligosaccharides, MS/MS was applied. Protonated molecular ions underwent extensive fragmentation, even under low-energy collision-induced dissociation. MS/MS spectra of [M+H]+ ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. Fragmentation of the [M+H]+ ions gave predominantly B- and Y-type glycosidic fragments. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could easily be obtained. Predictable and reproducible fragmentation patterns could be obtained in all cases. This derivatization procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from 10 microg of glycoproteins separated by gel electrophoresis. Moreover, the derivatives retain their sensitivity to exoglycosidases. Thus a series of sequential on-target exoglycosidase treatments combined with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was found to be useful for the determination of structural features of the glycans released from proteins separated by gel electrophoresis such as the monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. Our strategy can be used successfully to assign the major glycans released from proteins separated by gel electrophoresis.