ISSN:0173-0835    

DOI:10.1002/elps.1150180302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractMembrane and nuclear proteins of poor solubility have been separated by high resolution two‐dimensional (2‐D) gel electrophoresis. Isoelectric focusing with immobilized pH gradients leads to severe quantitative losses of proteins in the resulting 2‐D map, although the resolution is usually high. Protein solubility could be improved by using denaturing solutions containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and detergents (both nonionic and zwitterionic). The usefulness of thiourea‐containing denaturing mixtures is shown for microsomal and nuclear proteins as well as for tubulin, a protein highly prone to aggr

ISSN:0173-0835    

DOI:10.1002/elps.1150180303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThousands of proteins may be visualised on a two‐dimensional (2‐D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2‐D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub‐picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2‐D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the ‘reflux’ mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of spec

ISSN:0173-0835    

DOI:10.1002/elps.1150180304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA simple and inexpensive methacrylate rehydration chamber was built to accomodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory‐made strips were positioned in the grooves with the gel in contact with 500 μL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range ge

ISSN:0173-0835    

DOI:10.1002/elps.1150180305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractBasic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two‐dimensional (2‐D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4–10 and 6–10 using a previously published protocol (Görget al., Electrophoresis1988, 9, 531–546). In the present study we have extended the pH gradient to pH 12 with IPGs 8–12, 9–12 and 10–12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2‐D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady‐state conditions. Due to the selectivity of isoelectric focusing in IPG 9–12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone ext

ISSN:0173-0835    

DOI:10.1002/elps.1150180306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe reproducibility of complex protein patterns in two‐dimensional (2‐D) gels run with carrier ampholytes in the first dimension has been investigated. Two different laboratories collaborated in the study and 18 or 19 gels were run in each laboratory for comparison. The electrophoresis chemicals, running devices, and samples were standardized in both labs. The resulting 37 gels were scanned with a charge‐coupled device (CCD) camera and spots were located, counted, quantified, and matched using a commercially available image analysis system. Subsequently, the reproducibility of spot position was determined. To perform the statistical analysis, the test gels were initially each matched to a master references gel. Next, three sets of 12 gels (the image analysis software database could analyze only 12 gels at a time) were analyzed and the isoelectric point (pI) and molecular weight (Mr) positional variation of all the spots that matched across the gels in each set was determined. The resulting statistical analysis indicates very high reproducibility of the carrier ampholyte tech

ISSN:0173-0835    

DOI:10.1002/elps.1150180307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA simple theoretical model of low molecular mass ion adsorption on a weak exchanger is proposed. For a system containing immobilized charges in uniform concentration the pH change values connected with neutral salt addition and followed by further washing and voltage application are evaluated. The question of stability of the moving boundary, arising in the area near the electrodes, is considered for the constant current stabilization at different values of Immobiline concentration, pKvalues of Immoboline and salt concentration. The threshold values of ion mobility capable of providing a sharp salt front formation are also evaluated for conditions of strong adsorption. We found that, with extremely acidic Immobilines, a sharp salt front does not arise under the latter condition, even in the case of very high Immobiline concentrations.

ISSN:0173-0835    

DOI:10.1002/elps.1150180308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA number of proteins from a silver‐stained two‐dimensional (2‐D) electrophoresis gel of mouse liver whole‐cell lysate were identified by peptide mass mapping and sequene database searching. The excised protein spots were processed byin situreduction and alkylation, followed by Lys‐C digestion. The masses of the resulting peptide mixtures were measured with a matrix‐assisted laser desorption/ionization (MALDI) reflectron‐time‐of‐flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post‐source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spec

ISSN:0173-0835    

DOI:10.1002/elps.1150180309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractRecently, we have shown that a solid‐phase‐microextraction/capillary electrophoresis device coupled to an electrospray ionization triple quadrupole mass spectrometer through a microelectrospray interface represents a powerful analytical system for the rapid, conclusive and sensitive identification of proteins separated by gel electrophoresis. Here we report on the successful coupling of the same device to an electrospray ionization ion trap mass spectrometer and on the comparative evaluation of the performance of the triple quadrupole and ion‐trap‐based systems. In the ion trap mass spectrometer‐based system, using a tryptic digest of a calibrated bovine serum albumin sample, we achieved limits of detection in the single mass spectrometry (MS) and tandem MS mode, respectively, of 400 amol (if 20 μL of solution at a concentration of 20 amol/μL was applied). The system was also successfully used to identify six yeast proteins isolated from a single analytical two‐dimensional polyacrylamide gel. For the detection of unfragmented peptide ions both systems showed comparable sensitivity, whereas the ion‐trap‐based system showed superior performance with fra

ISSN:0173-0835    

DOI:10.1002/elps.1150180310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe success of the mass spectrometric‐based approaches for the identification of gel‐separated proteins relies upon recovery of peptides, without high levels of ionization‐suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to determine whether in‐gel or on‐membrane digestion provided a significant advantage when low to sub‐pmol quantities of gel‐separated proteins were analyzed by matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI‐MS) with respect to the number and size of released peptides. Serial dilutions of five standard proteins ofMr17 000 to 97 000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in‐gel digestion (using a microcolumn clean‐up protocol, Courchesne, P.L. and Patterson, S. D.,BioTechniques, 1997, in press) or on‐membrane digestion following blotting to the PVDF‐based membranes, Immobilon‐P and Immobilon‐CD. Peptide maps were able to be obtained for all proteins at the detection limit of each method (Immobilon‐P and Immobilon‐CD, 0.5 pmol; and in‐gel, 125 fmol), and searches of Swiss‐Prot or a non‐redundant database (>193 000 entries) successfully identified all of the proteins, except β‐casein. Fragment‐ion spectra using a curved‐filed reflector MALDI‐MS were obtained from more than one peptide per protein at loads down to 250 fmol (except β‐casein). Using the uninterpreted data, a search of the nonredundant database and a six‐way translation of GenBank dbEST (>2 208 000 entries total) was able to identi

ISSN:0173-0835    

DOI:10.1002/elps.1150180311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY