Articles
Rapid Fractionation Of Complex Protein Extracts With Centrifugal MembraneAdsorber Units Improves Detection Of Proteins In 2d Page
Wed, 05/19/2004 - 10:51am
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by M.K. Doud, M. W. Schmidt, D. A.Wolf, D. Hines, C. Naumann, A. Kocourek, N. Kashani-Poor and R. Zeidler
Introduction
Two-dimensional gel electrophoresis followed by mass spectrometry for protein identification is a reliable and reproducible means of sample analysis. A key element in this process is reducing sample complexity, as samples often consist of tens of thousands of proteins in a magnitude of different concentrations. While 2D gel electrophoresis has tremendous resolving power, a pre-fractionation step will enhance resolution, enabling more of the proteome to be visualized. Pre-fractionation can be achieved by protein extraction, purification of cell organelles, preparative isoelectric focusing (IEF), or chromatography, procedures that can be time consuming, result in sample loss and require expensive instrumentation.
Vivapure Ion Exchange Spin Columns (Vivascience, Carlsbad, Calif.), which employ a membrane adsorber technology as the chromatography matrix, were used to fractionate proteins based on differences in charge. Membrane adsorbers make use of convective transport to bring proteins to the ion exchange surface in microcentrifuge format. Binding, washing and elution steps are performed rapidly in standard equipment. The use of low binding materials together with centrifugal elution ensures outstanding sample recovery. In addition, the small, non-compressible bed reduces elution volume, eliminating the concentration step prior to acetone precipitation.
While this method should have application to a broad range of cell types, we demonstrate here its utility for increasing the resolving power of 2D PAGE based proteome analysis of the fission yeast Schizosaccharomyces pombe. In this example, the fission yeast cell lysate was applied to Vivapure Mini Q column and eluted in three fractions of increasing sodium chloride concentration. Each fraction, and the total lysate, were analyzd by 2D PAGE.
This simple pre-fractionation procedure considerably increases the resolving power of 2D-PAGE. Whereas 308 spots could be detected by analyzing total protein lysate, 910 spots were observed upon pre-fractionation. Thorough gel image analysis demonstrated that pre-fractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate.
Materials and methods
Preparation of cell lysate fission yeast cells (S. pombe cells DS 448-2) were grown at 30 C to a cell density of OD595 of 1.5. Then, cells were harvested, frozen at -80 C, and resuspended in lysis buffer (100 mM sodium bicarbonate pH 8.8, 0.5% triton + inhibitors PMSF, leupeptin and aprotinin). Cells were disrupted by bead lysis in a microcentrifuge tube. The lysate was applied to Vivapure Mini Q spin columns and eluted in fractions of increasing sodium chloride concentration (lysis buffer + NaCl) as described in detail below.
A Vivapure Q Mini spin column was equilibrated with 100 mM sodium bicarbonate buffer, pH 8.8 by loading 400 ml onto the column and spinning at 2000 3g for 5 min. 400 ml lysate (=4 mg) was loaded onto the equilibrated column and spun at 2000 3g for 5 min. Flow through was kept for further analysis (not shown). After binding, the spin column was washed 23 with 400 ml 100 mM sodium bicarbonate buffer, pH 8.8, then spun at 2000 3g for 5 min. The flow through was kept for further analysis (not shown in this report).
Fraction 1 eluted by loading 400 ml of a 100 mM sodium bicarbonate buffer, pH 8.8 containing 225 mM NaCl onto the column and spinning at 2000 3g for 5 minutes. Flow through was retained. (Fraction 1).
For the next elution step (Fraction 2) 400 ml of a 100 mM sodium bicarbonate buffer, pH 8.8 containing 300 mM NaCl was used as before.
For the last fractionation step (Fraction 3) 400 ml of a 100 mM sodium bicarbonate buffer, pH 8.8 containing 500 mM NaCl was used to elute the remaining proteins.
2D-PAGE analysis
All fractions, as well as the total lysate, were quantified and precipitated with TCA/acetone by adding 3 volumes of chilled (-20 C) 13.3 % TCA/0.093% 2-mercaptoethanol in acetone followed by incubation for 1.5 hours to overnight at -20 C. Samples were centrifuged at 5000 3g at -20 C, discarding the supernatants and re-suspending the pellets in chilled (-20 C) acetone, containing 0.07% 2-ME to remove all of the TCA. Samples were spun down again at 5000 3g at -20 C. All acetone was removed and the pellets left to dry at 30 C. The pellets were then re-dissolved in CHAPS buffer (7M Urea, 2M thio-urea, 2% CHAPS, 2% ampholytes 3-10, 65 mM DTT, 0.1% bromophenol blue) to a concentration of 2.5 mg/ml. 400 ml (1 mg) of this was loaded onto a IPG strip (18 cm pH 3-10 NL IPG strip, (Amersham Biosciences, Piscataway, NJ)) for IEF continuing with 2D PAGE (22 cm 3 22 cm 8-18% Duracryl SDS PAGE gel (Genomic Solutions, Ann Arbor, Mich.). The procedure is outlined in Figure 1.
Results and discussion
To visualize more of the fission yeast proteome, a simple and reproducible method to prefractionate cell lysate using Vivapure anion exchange spin columns was sought. 4 mg total cell protein was applied to the column in a volume of 400 ml and eluted with increasing concentrations of sodium chloride in increments of 50 mM. Figure 2a summarizes the binding and elution characteristics of the spin column. Individual fractions were run on a 1D PAGE gel to determine suitable fraction pooling for 2D PAGE. Figures 2a and 2b provide elution profile and gel results. This resulted in 225 mM, 300 mM, and 500 mM NaCl concentrations being chosen for elution. The eluates were precipitated and separated using IEF in the first dimension and then using PAGE (Figure 3).
Visually, differences in spot allocation and resolution could be detected comparing the different fractions and the original lysate. Detailed software analysis showed that most of the spots (Figure 4) resolved in the NaCl elution fractions could not be found in the original lysate (blue circles). For example, a zoom image for a section of 2D gel achieved with Fraction 3 (elution with 500 mM NaCl) shows a gel region containing more spots than detectable in the original lysate.
A complete quantification of the spots is shown in Table 1.
Conclusions
Pre-fractionation with centrifugal membrane adsorber units is a simple and reliable method to increase the resolving power of 2D-PAGE. Pre-fractionation led to a threefold increase in the number of features discernible by 2D-PAGE. Approximately 50% of these features are uniquely represented in the fractions in contrast to the total protein lysate.
About the authors
D. A. Wolf, M.K. Doud and M. W. Schmidt are at the Harvard NIEHS Center Proteomics Facility, Dept. of Genetics and Complex Diseases, Harvard School of Public Health. D. Hines is with Vivascience Inc. in Carlsbad, Calif., and C. Naumann, A. Kocourek, N. Kashani-Poor and R. Zeidler are with Vivascience AG in Hannover, Germany. More information about techniques discussed is available from: Vivascience, Inc.
Use InfoLINK 4E1701 or Call 800-287-0633
Figure 1. Workflow for fractionation of yeast cell lysates using Vivapure Q spin columns by applying increasing NaCl concentrations for elution. Figure 2a. 4 mg total yeast protein was loaded onto a Vivapure Q mini spin column, washed and eluted with a NaCl step gradient from 50 mM to 1 M. Most of the protein could be eluted around 200-300 mM NaCl. Figure 2b. Analysis of fractions on 1D PAGE. Flow though as well as high- and low-salt fractions showed different protein patterns. |
Two-dimensional gel electrophoresis followed by mass spectrometry for protein identification is a reliable and reproducible means of sample analysis. A key element in this process is reducing sample complexity, as samples often consist of tens of thousands of proteins in a magnitude of different concentrations. While 2D gel electrophoresis has tremendous resolving power, a pre-fractionation step will enhance resolution, enabling more of the proteome to be visualized. Pre-fractionation can be achieved by protein extraction, purification of cell organelles, preparative isoelectric focusing (IEF), or chromatography, procedures that can be time consuming, result in sample loss and require expensive instrumentation.
Vivapure Ion Exchange Spin Columns (Vivascience, Carlsbad, Calif.), which employ a membrane adsorber technology as the chromatography matrix, were used to fractionate proteins based on differences in charge. Membrane adsorbers make use of convective transport to bring proteins to the ion exchange surface in microcentrifuge format. Binding, washing and elution steps are performed rapidly in standard equipment. The use of low binding materials together with centrifugal elution ensures outstanding sample recovery. In addition, the small, non-compressible bed reduces elution volume, eliminating the concentration step prior to acetone precipitation.
While this method should have application to a broad range of cell types, we demonstrate here its utility for increasing the resolving power of 2D PAGE based proteome analysis of the fission yeast Schizosaccharomyces pombe. In this example, the fission yeast cell lysate was applied to Vivapure Mini Q column and eluted in three fractions of increasing sodium chloride concentration. Each fraction, and the total lysate, were analyzd by 2D PAGE.
This simple pre-fractionation procedure considerably increases the resolving power of 2D-PAGE. Whereas 308 spots could be detected by analyzing total protein lysate, 910 spots were observed upon pre-fractionation. Thorough gel image analysis demonstrated that pre-fractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate.
Materials and methods
Preparation of cell lysate fission yeast cells (S. pombe cells DS 448-2) were grown at 30 C to a cell density of OD595 of 1.5. Then, cells were harvested, frozen at -80 C, and resuspended in lysis buffer (100 mM sodium bicarbonate pH 8.8, 0.5% triton + inhibitors PMSF, leupeptin and aprotinin). Cells were disrupted by bead lysis in a microcentrifuge tube. The lysate was applied to Vivapure Mini Q spin columns and eluted in fractions of increasing sodium chloride concentration (lysis buffer + NaCl) as described in detail below.
A Vivapure Q Mini spin column was equilibrated with 100 mM sodium bicarbonate buffer, pH 8.8 by loading 400 ml onto the column and spinning at 2000 3g for 5 min. 400 ml lysate (=4 mg) was loaded onto the equilibrated column and spun at 2000 3g for 5 min. Flow through was kept for further analysis (not shown). After binding, the spin column was washed 23 with 400 ml 100 mM sodium bicarbonate buffer, pH 8.8, then spun at 2000 3g for 5 min. The flow through was kept for further analysis (not shown in this report).
Fraction 1 eluted by loading 400 ml of a 100 mM sodium bicarbonate buffer, pH 8.8 containing 225 mM NaCl onto the column and spinning at 2000 3g for 5 minutes. Flow through was retained. (Fraction 1).
For the next elution step (Fraction 2) 400 ml of a 100 mM sodium bicarbonate buffer, pH 8.8 containing 300 mM NaCl was used as before.
For the last fractionation step (Fraction 3) 400 ml of a 100 mM sodium bicarbonate buffer, pH 8.8 containing 500 mM NaCl was used to elute the remaining proteins.
Figure 3. Shows 2D PAGE of original lysate and 225 (Fraction 1), 300 (Fraction 2) and 500 (Fraction 3) mM NaCl elutions. Figure 4. Analysis of the 500 mM fraction (Fraction 3). A comparison between fraction 3 and the original lysate demonstrate the detection of additional protein spots within the circled region. |
All fractions, as well as the total lysate, were quantified and precipitated with TCA/acetone by adding 3 volumes of chilled (-20 C) 13.3 % TCA/0.093% 2-mercaptoethanol in acetone followed by incubation for 1.5 hours to overnight at -20 C. Samples were centrifuged at 5000 3g at -20 C, discarding the supernatants and re-suspending the pellets in chilled (-20 C) acetone, containing 0.07% 2-ME to remove all of the TCA. Samples were spun down again at 5000 3g at -20 C. All acetone was removed and the pellets left to dry at 30 C. The pellets were then re-dissolved in CHAPS buffer (7M Urea, 2M thio-urea, 2% CHAPS, 2% ampholytes 3-10, 65 mM DTT, 0.1% bromophenol blue) to a concentration of 2.5 mg/ml. 400 ml (1 mg) of this was loaded onto a IPG strip (18 cm pH 3-10 NL IPG strip, (Amersham Biosciences, Piscataway, NJ)) for IEF continuing with 2D PAGE (22 cm 3 22 cm 8-18% Duracryl SDS PAGE gel (Genomic Solutions, Ann Arbor, Mich.). The procedure is outlined in Figure 1.
Results and discussion
To visualize more of the fission yeast proteome, a simple and reproducible method to prefractionate cell lysate using Vivapure anion exchange spin columns was sought. 4 mg total cell protein was applied to the column in a volume of 400 ml and eluted with increasing concentrations of sodium chloride in increments of 50 mM. Figure 2a summarizes the binding and elution characteristics of the spin column. Individual fractions were run on a 1D PAGE gel to determine suitable fraction pooling for 2D PAGE. Figures 2a and 2b provide elution profile and gel results. This resulted in 225 mM, 300 mM, and 500 mM NaCl concentrations being chosen for elution. The eluates were precipitated and separated using IEF in the first dimension and then using PAGE (Figure 3).
Visually, differences in spot allocation and resolution could be detected comparing the different fractions and the original lysate. Detailed software analysis showed that most of the spots (Figure 4) resolved in the NaCl elution fractions could not be found in the original lysate (blue circles). For example, a zoom image for a section of 2D gel achieved with Fraction 3 (elution with 500 mM NaCl) shows a gel region containing more spots than detectable in the original lysate.
A complete quantification of the spots is shown in Table 1.
| Fraction 1 | 307 spots on gel | Lysate: 308 spots on gel |
| 93 match Lysate | ||
| 33 match Fraction 2 | ||
| 25 match Fraction 3 | 156 Unique | |
| Fraction 2 | 302 spots on gel | |
| 58 match Lysate | ||
| 33 match Fraction 1 | ||
| 36 match Fraction 3 | 175 Unique | |
| Fraction 3 | 283 spots on gel | |
| 95 match Lysate | ||
| 25 match Fraction 1 | ||
| 36 match Fraction 2 | 127 Unique | |
Pre-fractionation with centrifugal membrane adsorber units is a simple and reliable method to increase the resolving power of 2D-PAGE. Pre-fractionation led to a threefold increase in the number of features discernible by 2D-PAGE. Approximately 50% of these features are uniquely represented in the fractions in contrast to the total protein lysate.
About the authors
D. A. Wolf, M.K. Doud and M. W. Schmidt are at the Harvard NIEHS Center Proteomics Facility, Dept. of Genetics and Complex Diseases, Harvard School of Public Health. D. Hines is with Vivascience Inc. in Carlsbad, Calif., and C. Naumann, A. Kocourek, N. Kashani-Poor and R. Zeidler are with Vivascience AG in Hannover, Germany. More information about techniques discussed is available from: Vivascience, Inc.
Use InfoLINK 4E1701 or Call 800-287-0633
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