Rapid prefractionation of complex protein lysates with centrifugal membrane adsorber units improves the resolving power of 2D-PAGE-based proteome analysis
© Doud et al; licensee BioMed Central Ltd. 2004
Received: 20 April 2004
Accepted: 26 April 2004
Published: 26 April 2004
Two-dimensional gel electrophoresis (2D-PAGE) has proven over the years to be a reliable and efficient method for separation of hundreds of proteins based on charge and mass. Nevertheless, the complexity of even the simplest proteomes limits the resolving power of 2D-PAGE. This limitation can be partially alleviated by sample prefractionation using a variety of techniques.
Here, we have used Vivapure Ion Exchange centrifugal adsorber units to rapidly prefractionate total fission yeast protein lysate based on protein charge. Three fractions were prepared by stepwise elution with increasing sodium chloride concentrations. Each of the fractions, as well as the total lysate, were analyzed by 2D-PAGE. This simple prefractionation procedure considerably increased the resolving power of 2D-PAGE. Whereas 308 spots could be detected by analysing total protein lysate, 910 spots were observed upon prefractionation. Thorough gel image analysis demonstrated that prefractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate.
Prefractionation with Vivapure Q spin columns proved to be a simple, fast, reproducible, and cost-effective means of increasing the resolving power of 2D-PAGE using standard laboratory equipment.
Despite some limitations, 2-dimensional gel electrophoresis (2D-PAGE)  coupled to mass spectrometric protein identification remains one of the most reliable and reproducible means of proteome analysis. Due to the wide dynamic range of individual proteins in cells, which presumably varies over five to six orders of magnitude , a key requirement for a comprehensive proteome analysis is to reduce sample complexity to a level that permits access to low abundance proteins. Whereas the resolving power of 2D-PAGE is remarkable, biochemical prefractionation will further enhance resolution enabling a deeper view into complex proteomes (reviewed in ).
Prefractionation can be achieved by a number of techniques such as differential protein extraction, purification of cell organelles or protein complexes, preparative isoelectric focusing (IEF), or chromatographic techniques . Many of these procedures are time consuming, difficult to reproduce and scale up, result in sample loss, and often require expensive instrumentation such as liquid chromatography systems.
Here, we have tested Vivapure Ion Exchange Spin Columns employing a membrane adsorber technology as the chromatography matrix to fractionate proteins based on differences in charge. Unlike traditional chromatography resins, membrane adsorbers make use of convective transport to bring proteins to the ion exchange surface in microcentrifuge format. Hence, the binding, washing, and elution steps are performed rapidly in standard laboratory equipment. Moreover, the use of low binding materials together with centrifugal elution ensures highly efficient sample recovery.
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.
Results and discussion
Fig. 3B shows the elution profiles of two independent samples. The nearly complete overlap of the two curves indicated that the prefractionation and step elution was highly reproducible. In addition, the three fractions contained a similar amount of protein (~0.5 mg). The 2D-PAGE analysis showed that prefractionation enriched for many proteins that were not visible in unfractionated lysate (Fig. 3C). Whereas 308 spots were visible in the total cell lysate, 307, 302, and 283 spots were observed in fractions 1, 2, and 3, respectively (Fig. 3C and Table 1). In addition, as expected for successful fractionation by charge, there was a successive enrichment of more acidic proteins as the NaCl concentration of the elution buffer increased (Fig. 3C). Overall, the spin column prefractionation led to a similar deconvolution of sample complexity as we had previously observed with anion exchange chromatography on an automated FPLC system .
Prefractionation with centrifugal membrane adsorber units was found to be a simple and reliable method to increase the resolving power of 2D-PAGE. Prefractionation led to a threefold increase in the number of features descernible by 2D-PAGE. Approximately 50% of these features are uniquely represented in the fractions, but not in total protein lysate.
Preparation of total cell lysate
A liquid culture of 50 ml fission yeast cells (927 h- leu1-32 ura4-d18) was grown at 30°C to an OD595 of 1.5. Cells were harvested by centrifugation, frozen at -80°C, and resuspended in 500 ul lysis buffer (100 mM sodium bicarbonate pH 8.8, 0.5% Triton X100, and protease inhibitors (1 mM PMSF, 10 ug/ml leupeptin, 10 ug/ml pepstatin, 15 ug/ml aprotinin)). Cells were disrupted by bead lysis in a microfuge tube containing 0.5 ml Zirconia beads (0.5 mm, Biospec Products. Inc.) and the cell debris was removed by centrifugation for 15 minutes at 4°C. The protein concentration was determined with the BioRad DC Protein Assay using BSA as a standard. 1 ml protein lysate (~10 mg/ml) was incubated with 100 ug/ml RNAseA and DNAseI for 15 minutes on ice and cleared by ultracentrifugation at 395,000 × g for 30 minutes at -20°C.
Prefractionation on membrane adsorber units
A Vivapure Q Mini spin column was equilibrated with 100 mM sodium bicarbonate buffer, pH 8.8 by loading 400 μl onto the column and spinning at 2000 × g for 5 minutes. 400 ul lysate (= 4 mg protein) was loaded onto the equilibrated spin column and spun at 2000 × g for 5 minutes. After binding, the column was washed twice with 400 ul 100 mM sodium bicarbonate, pH 8.8. Fraction 1 was eluted by loading 400 ul 100 mM sodium bicarbonate, pH 8.8 containing 225 mM NaCl onto the column and spinning at 2000 × g for 5 minutes. Subsequent elutions were performed with 400 ul 100 mM sodium bicarbonate buffer, pH 8.8, 300 mM (fraction 2) or 500 mM NaCl (fraction 3), respectively.
Fractions, as well as the total lysate, were quantified and then precipitated by adding 3 volumes of chilled (-20°C) 13.3 % TCA / 0.093% 2-mercaptoethanol in acetone, followed by incubation overnight at -20°C. Samples were centrifuged at 5000 × g at -20°C and pellets were resuspended in chilled (-20°C) acetone, containing 0.07% 2-mercaptoethanol. Samples were spun again at 5000 × g at -20°C. All acetone was removed and the pellets left to dry at 30°C. The pellets were then redissolved at 30°C in CHAPS buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2% ampholytes 3–10, 65 mM DTT, 0.1% bromophenol blue) to a concentration of 2.5 mg/ml. 400 ul (1 mg) of this was loaded onto an 18 cm immobilized pH 3–10 nonlinear gradient strip (Amersham) and passively rehydrated for 16 hours. The strips were then focused to 100,000 Vh (Genomic Solutions Investigator), equilibrated in 10 ml equilibration buffer I (6 M urea, 375 mM Tris/HCl pH 7.4, 2% SDS, 2% glycerol, 2% DTT), followed by 10 ml equilibration buffer II (6 M urea, 375 mM Tris/HCl pH 7.4, 2% SDS, 2% glycerol, 2% iodoacetamide), and applied to an 8–18% gradient Duracryl SDS PAGE gel (Genomic Solutions). The gels were stained with SYPRO Ruby and imaged on a BioRad Molecular Imager FX Pro Plus (532 nm excitation, 555 nm emission filter, 1064 nm excitation filter).
The images were analyzed using Phoretix 2D software from Nonlinear Dynamics. Spots were detected using automatic spot detection and the background was subtracted using the mode of non-spot method. The images were also subjected to spot filtering in order to remove spots occurring as a result of residual dye crystals. The three fractions were matched against the total lysate by placing thirty seed matches from which automatic matches were then made. The three fractions were also matched against one another by the same method to determine the number of unique spots in each fraction.
List of abbreviations
two-dimensional polyacrylamide gel electrophoresis
sodium dodecyl sulfate
The authors thank Drs. Reif, Nussbaumer, and Demmer (Sartorius AG, Göttingen, Germany) for their support and assistance regarding Vivapure Ion Exchange membrane adsorbers. This work was funded by the NIEHS Center grant ES00002 and by NIH grant RO1-GM9780 to DAW.
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