Anal. Chem. 1994,66, 2529-2536
Two-Dimensional Gel Electrophoresis/Liquid Chromatography for the Micropreparative Isolation of Proteins Donald J. Rose’ and Gregory J. Oplteckt Department of Bioanalytical and Structural Chemistty, Glaxo, Inc. Research Institute, 5 Moore Drive, Research Triangle Park, North Carolina 27709
Although comprehensive, column-based two-dimensional separation techniquesoffer enormous resolving power for a complex mixture, they often lack the ability to isolate the separated species for further analysis (e.g., proteins for Edman sequencing). This paper describes a micropreparative two-dimensional separation system for the isolation of proteins from complex mixtures, such as cell lysates. The system is composed of commerciallyavailableequipment: continuous-elutiontube gel electrophoresis as the first dimension followed by gradient elution, reversed-phaseperfusionliquid chromatographyas the second dimension with a two-loopsampling valve as the interface between dimensions. The two-dimensional electrophoresis/ liquid chromatography system (ZD-EP/LC) shows high resolution of proteins since each dimension has orthogonal separation mechanisms (electrophoresis, size/charge; LC, hydrophobicity). Identificationof proteinsfor further analysis is accomplished by superimposing a grid on the computergenerated 3D image. For the analysis of complex mixtures, for example, components in a biological sample, a separation technique with high resolving is essential. However, the use of a single separation technique is usually insufficient to obtain adequate resolution. One approach to increasing resolutionis to combine two separation techniques into a two-dimensional separation system. The rationale for two-dimensional separation techniques comes from the work of Davis and Gidding~,l-~ where they point to the limitations of one-dimensional chromatographic separations of complex mixtures due to the statistical theory of multicomponent overlap. The theoretical work on two-dimensional separations, developed by Giddings,4.5 shows that when two orthogonal separation techniques are coupled, the resulting peak capacity is the product of the individual technique’s peak capacities. Excellent reviews of the theoretical and experimental basis of two-dimensional separations have been published recently.”8 Current address: Department of Chemistry, University of North Carolina, Chapel Hill, N C 27599. ( I ) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983, 55, 418-424. (2) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2168-2177. (3) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2178-2182. (4) Giddings, J. C. Anal. Chem. 1984, 56, 1258A-1270A. (5) Giddings,J. C. HRCCC, J. High Resolut. Chromatogr.Chromatogr.Commun. 1987, 10, 319-323. (6) Lemmo, A. V.;Larmann, J. P.; Moore, A. W.; Jorgenson, J. W.; Liu, Z.; Lee, M. L. Anal. Chem. in preparation. (7) Cortes, H. J. J. Chromatogr. 1992, 626, 3-23. (8) Cortes, H. J. Multidimemionalchromatography:techniques and applicationr; Marcel Dekker: New York. 1990. 0003-2700/94/0366-2529$04.50/0
0 1994 American Chemical Society
Experimentally, two-dimensional separations involve a second dimension separation technique sampling the effluent from the first dimension separation. One approach to twodimensional separations involvesdiverting a single, unresolved peak from the first dimension into a second separation technique . Such “heartcutting” methods have a limited number of samplings of the first dimension and require preliminary runs to determine the appropriate time to sample the first dimension. In contrast to the single sampling of the heartcutting techniques, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) performs infinite samplings of the first dimension by separating components according to charge in an isoelectric focusing gel in the first dimension and then physically laying the gel containing the components on the edge of a slab gel to separate in the second dimension according to size in an SDS-PAGE gel. This type of “comprehensive” two-dimensional separation, where all the components of the first dimension are separated in the second dimension, provides the most resolution and peak capacity and, thus, the most analytical information for an unknown mixture. However, to achieve this level of sampling of the first dimension requires manual manipulation of the gel and results in reproducibilityand quantitation inferior to that found in column-based separations methods such as liquid chromatography and capillary electrophoresis. To realize the high resolving power and peak capacity of 2D-PAGE in an automated, column-based format, the effluent from the first dimension must be discretely sampled by the second dimension such that the sampling frequency is sufficient to retain any resolution achieved in the first dimension. One type of comprehensive two-dimensional separation directs the entire effluent from the first dimension into the second dimension, without loss of sample. In this case, the first dimension effluent stream can be segmented by either simply stopping the flow of the first dimension after each samplinggJ0 or using a switching valve with a pair of sample loops such that effluent from the first dimension fills one sample loop while the second loop is being injected and separated in the second dimension.”J2 In the latter case, the volumetric flow rate of the first dimension effluent must be compatible with the injection volume and run time for the second dimension. (9) Matsuoka, K.; Toaka, M.; Isobe, T.; Okuyama, T. J. Chromatogr. 1990,515,
3 13-320. (10) Takahashi, N.; Takahashi, Y.; Putnam, F. W. J. Chromatogr. 1983, 266, 51 1-522. (1 1) Erni, F.; Frei, R. W. J. Chromatog. 1978, 149, 561-569. (12) Bushcy, M. M.; Jorgcnson, J. W. AMI. Chem. 1990,62, 161-167.
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HPLC column
i collector
itex support membrane
Flgure 1. Schematic of two-dimensional electrophoresls/liquM chromatography (SD-EP/LC) separation system. 1.2
For example, if the first dimension flow rate is x mL/min and maximum injection volume for the second dimension is y mL, then the run time for the second dimension must be no greater thanylx minutes. With theadvent of low flow-rate separation methods such as microbore or packed capillary liquid chromatography and fast chromatographic methods such as perfusion chromatography, compatibilitycan be easily achieved. For example, in comprehensive two-dimensional liquid chromatography/liquid chromatography (2D LC/LC),12 highspeed size-exclusion chromatography as the second dimension (6 min/run) can accept the 3O-wL injection volume from a microbore LC flowing at 5 pL/min. The second type of comprehensive two-dimensional separation involves segmentation of the first dimension effluent stream using sampling loops, as described above, but only a portion of each segment is injected into the second dimension. This allows coupling of techniques such as packed capillary LC and capillary e l e c t r o p h ~ r e s i s , ~where ~ - ~ ~the segmented volume from the first dimension (microliters) is much greater than the acceptable injection volume of the second dimension (nanoliters). Previously, the high resolving power of comprehensive 2D separations had been primarily used as an analytical mapping or “fingerprinting” tool for complex mixtures. Because of the small scale of the separation system, the total amount of component eluting from the second dimension is insufficient to collect and analyze. A micropreparative two-dimensional separation would permit the isolation of proteins from complex mixtures which could be characterized further by Edman sequencing or mass spectrometry. In designing a micropreparative 2D system, the first requirement is that enough sample be loaded in the first dimension so that, after dilution from the second dimension, the concentration is still sufficient to detect and collect. Micropreparative tube gel electro(13) Lemmo, A. V.; Jorgenson, J. W. J . Chromatogr. 1993,633,213-220. (14) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993,65,15761581. (15) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62,978-984.
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AnalvtlcaIChemlstry, Vol. 66, No. 15, August 1, 1994
void volume
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Figure 2. One of the onedimensional H R C separations of protein standards used to construct PD-EPlLC Image shown in Figure 3 (run in Figure 3). Condltions: see Figure 3. taken at 124 min, ‘9”
phoresis with continuous elution meets this requirement, since it can handle hundreds of micrograms of protein. The second requirement is that the two-dimensional separation be comprehensive (Le., all sample components pass through both dimensions) to provide resolution of all components of an unknown mixture as well as to ensure the most protein collected after the second dimension. To meet this requirement, the second dimension analysis, as shown in other comprehensive 2D work,12needs to be rapid enough to allow sufficiently high sampling frequency of the first dimension effluent. Furthermore, the volume sampled from the first dimension should not exceed the maximum injection volume or the sample capacity of the second dimension, otherwise partial injection of the sampled volumeswill be necessary,14reducing the overall sensitivity of the separation. With introduction of high-speed chromatographic packing materials such as perfusive media,16 fast, second dimension LC runs can be accomplished with (16) Afeyan, N. B.; Gordon, N. F.; Mazsaroff, I.; Varady, L.; Fulton, S.P.; Yang, Y.B.; Regnier, R. E. J. Chromatogr. 1990, 519, 1-29.
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HPLC Time (min) Flgure 3. 2D-EPILC separation of standard proteins lactalbumin, bovine serum albumin, ovalbumin, carbonic anhydrase, horse muscle myoglobin, and transferrin. Conditions: gel column, 2.5 X 50 mm, 7.5% T; elution buffer flow rate, 25 pL/min; electrophoresis, from 0.2 to 0.8 mA in 10 min, 0.8 mA for 320 min; sampling loops, 100 pL; HPLC, 2.1 X 100 mm POROS R/H 11, from 30 to 50% acetonitrile in 3.5 min, 0.5-min reequilibration (3.2 column volumes), 1.5 mL/min; sample, 20 pL of 0.83 pg/mL per protein (17 pg/protein, 380 pmoi for ovalbumin). The Intensity of the spots represents absorbance at 2 10 nm (red, highest absorbance; purple, lowest absorbance).
little loss in resolution. Furthermore, using gradient elution LC, as compared to isocratic elution, one can accommodate a wider range of injection volumes while concentrating the sample before separation. This paper describesa unique twodimensional separation systemcomprising continuous-elution tube gel electrophoresisas the first dimension coupled to highspeed gradient elution, reversed-phase perfusion liquid chromatography. The system is characterized in terms of the first and second dimensions and applied to the separation, collection, and analysis of components in complex biological samples.
EXPERIMENTAL SECTION Apparatus. The two-dimensional electrophoresis/liquid chromatography separation system (2D-EP/LC), shown in Figure 1, is composed of a continuous-elution tube gel electrophoresisinstrument (230A, Applied Biosystems, Foster City, CA) coupled to a reversed-phaseHPLC system (pump, 1090, Hewlett-Packard, Palo Alto, CA; detector, UVis 200, Linear Instruments, Reno, NV; flow cell, 6-mm path length, 9-pL volume; fraction collector, SF-2 120, Advantec, Dublin, CA) using an electronically actuated eight-port switchingvalve as the interface between dimensions (E45/C8W, Valco, Houston, TX). The electrophoresis instrument contains a native polyacrylamide gel tube column mounted between two reservoir blocks, each of which provides electrical contact as well as a continuous flow of buffer during electrophoresis. The blocks and gel electrophoresis tube are housed in Peltiercooled chamber to cool the gel tube during electrophoresis.
The lower block (inset, Figure 1) is divided into two compartments by a low molecular weight dialysis membrane: the lower compartment continuously flushed with lower reservoir buffer and the upper compartment pumped with elution buffer toward the switching valve using a syringepump (140B, ABI). During electrophoresis, low molecular weight ions pass out of the bottom of the gel and through the membrane toward the positive (grounded) electrode while large molecular weight proteins are retained in the upper compartment and swept out of the block with elution buffer. The eight-port switchingvalve is equipped with two matched sampling loops (25-100 pL) which act as the injection loops for the HPLC. The flow rate of the elution pump and size of the sample loops determine the run time for the HPLC (e.g., an elution flow rate of 25 pL/min with 100-pLsampling loops requires a 4-min HPLC run time). The fraction collector is custom programmed (Advantec) to allow the collection of 20 HPLC runs, each divided into 17 fractions of up to 1 mL each (340 fractions total). In addition, the order of the fraction collection has been changed from serpentine (down one row and up the next) to row by row (down each row in the same direction). This movement produces a tray of fractions that can be correlated to the 3D plot. The 2D-EP/LC system is controlled by a microcomputer (Macintosh IIfx, Apple Computer, Cupertino, CA) running LabView and equipped with two multifunction 1/0 boards (NB-MIO- 16and DMA-2800,National Instruments,Austin, TX). The computer interface controls the syringepump (RSAnalytical Chemistty, Vol. 66, No. 15, August 1, 1884
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Figure 4. PD-EPILC separation of standard proteins on a (A) 2.5 mm i.d. or (B) 1.0 mm i.d. X 50 mm long gel column. Conditions: see Figure 3.
232/422), senses the start of the electrophoresis run (digital I/O), actuates the switching valve (relay contact closure), controls the HPLC gradient and flow rate (GPIB), collects the HPLC UV absorbance data (A/D conversion), and advances the fraction collector (relay contact closure). 2D data collected by LabView (absorbancefrom a series of HPLC runs) is stored and viewed one HPLC run at a time or exported as ASCII data to Transform (Spyglass, Champaign, IL) for creation of 3D plots. Materials. The gel tube columns (1 or 2.5 mm i.d. X 50 mm) for the first dimension separation contained either 5 or 7.5% T, 2.6% C native polyacrylamidegel and were prepared and stored according to the manufacturer’s protocol (% T is the total monomer weight per 100 g of buffer solution and % C is the percent cross-linker of total monomer). Lactalbumin (MW 14 000, no p l available), ovalbumin (MW 45 000, p l 4.7), bovine serum albumin (MW 66 000, pl4.9), carbonic anhydrase (29 000, p l 5.9), horse muscle myoglobin (MW 17.5 000, PI6.5), and transferrin (MW 72 000, pl5.9) were purchased from Sigma (St. Louis, MO). Cell lysates were prepared at Glaxo. The buffers used for electrophoresis were according to the manufacturer’s protocol for native protein gel electrophoresis and included the following: gel buffer, 375 mM Tris-HC1, pH 8.8; upper buffer, 25 mM Tris, 192 mM glycine, pH 8.8; lower buffer, 25 mM Tris-HC1, pH 8.3; elution buffer, 50 mM Tris-HC1, pH 8.3; sample buffer, 20 mM Tris-HC1, pH 9.5,15% glycerol. The second dimension reversed-phase HPLC column (POROS R / H I1 2.1 mm X 100 mm or 30 mm, Perseptive Biosystems, Cambridge, MA) used a gradient mobile phase mixed between two solvents, 0.1% TFA in water and 0.1% TFA in acetonitrile. Procedure. A gel column was removed from the storage buffer, fitted with a Zitex membrane on either end to prevent gel extrusion, loaded into the electrophoresis blocks, and run at constant current for 150 min to eliminate polymerization byproducts (from 0.2 to 0.8 mA over 30 min and 0.8 mA for 120 min, typically reaching 175 V). After this preelectrophoresis, a native protein sample in sample buffer (-20 pL) was applied to the top of the gel tube and electrophoresed at constant current for 2-4 h (from 0.2 to 0.8 mA for 10 min and 0.8 mA thereafter). During electrophoresis, the upper and lower reservoir buffers flowed at -2 mL/min and the 2532
Analytical Chemistry, Vol. 66, No. IS,August 1, 1994
elution buffer flowed at 25 pL/min. After a specified delay time (the time for a tracking dye to migrate the length of the tube, typically 40 min), the switching valve was rotated to position A, injecting the volume of loop A (typically 100 pL, see Figure 1) into the HPLC column. The proteins from the injection volume were concentrated by using an initially low (20%) organic mobile phase. After injection, the components were eluted from the column using a gradient mobile phase (typically from 20 to 50% acetonitrile in 3.5 min at 1.5 mL/ min), and the column was reequilibrated with the initial gradient conditions (1.5 mL/min for 0.5 min, approximately three column volumes). Eluted componentswere detected at 210 nm and collected, if desired. During the HPLC run of the contents of loop A, loop B was being filled with eluent from the electrophoretic separation. Thus, at the end of the HPLC run, the valve was rotated to position B, injecting loop B, the next segment of elution buffer from electrophoretic separation. Likewise, as the components from loop B were separated, loop A was filled with electrophoretic eluent. Rotation of the valve between positions A and B continued for the duration of the electrophoresis run (90-360 min). A series of HPLC runs (1D runs), one of which is shown in Figure 2, were plotted along the same HPLC time axis, interpolated, and rendered in a false color map to produce a 3D image of the 2D separation space, as shown in Figure 3 (electrophoresis time X HPLC time X HPLC absorbance). If fractions were collected, a grid pattern was superimposed on the image to determine which fractions were to be used for further analysis. RESULTS AND DISCUSSION First Dimension. Continuous-elution tube gel electrophoresis offers several advantages as the first dimension separation. First, because the gel electrophoresis is an electricfield-driven separation method, as opposed to fluid-driven methods such as HPLC, the flow rate from the first dimension into the second dimension is independentof the first dimension separation (A lower limit of 10pLlmin sample elution flow must be maintained for efficient removal of electrolysis products from the elution chamber.) Decoupling of the flow rate from the separation permits flexibility in terms of column dimensions,gel porosity, and sampleload of the first dimension.
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F h r e 5. PD-EPILC separation of standard proteins using either (A) 50- or (B) 250-pL sampling loops. Distance between white lines shows the width of one HPLC run. Conditions: same as Figure 3 except for (A) 2.1 X 30 mm POROS R/H, from 30 to 50 % acetonitrile gradient in 1.8 min, O.2-min reequilibration (9.3 column volumes), 2.8 mL/min; (B) 2.1 X 100 mm POROS R/H ,from 30 to 50 % acetonitrile in 9 min, 1-min reequilibration (6.5 column volumes), 1.5 mL/min.
Second, the separation mechanism for native gel electrophoresis is orthogonal to that of most types of HPLC. Native gel electrophoresis is a multimode separation method, separating proteins by both charge and size; the contribution of the two mechanisms is determined by the gel porosity and pH. Finally, a load of up to several hundred micrograms of protein can be applied to the gel, which, after dilution effects of the second dimension HPLC, is sufficient to detect, collect, and analyze further. However, the use of gel electrophoresisas the first dimension separation imposes some limitations. For example, because the polyacrylamidegel has limited stability above pH 9, basic proteins (PI > 9) have a prohibitively low mobility through the gel tube (If only highly basic proteins are to be analyzed, acidicgels with a reversed polarity could be used). Compared to HPLC columns, polyacrylamide gel columns are not as reproducible from run to run and between columns, due mainly to degradation of the polyacrylamide matrix with time and irreproducibility of the polymerization reaction. For example, the run-to-run reproducibility of migration time for the test protein lactalbumin was 8% RSD (n = 7). In general, only five to seven runs could be conducted with a newly prepared gel column before significant losses in resolution occurred (Storedcolumns had less longevity.) Even though the columns can be made in batches, each new column requires a 2-3 h preelectrophoresis to remove polymerizationbyproducts, and the storage life of each column is limited to weeks. Resolution (Rs)in electrophoresis can be expressed in the following terms:"
where X is the migration distance q is the average charge of the species, E is the electric field, R is the gas constant, T is the absolute temperature, P u i s the velocity differencebetween the two species, and Uis the average velocity. One approach to improving resolution involves manipulating the pH of the (17) Gidding, J. C. Sep. Sci. 1963.4, 181-189.
gel buffer to affect a change the net charge and thus the net mobility of the protein. Since most of the samples dealt with in this work were of unknown protein composition and since rapid analysis time was more important than optimum resolution, gel columns were run at the highest pH possible (pH 8.8) without significant degradationof the polyacrylamide matrix. Changes in gel concentration (% T) can improve resolution1*J9by changing the relative mobilities as sieving plays a larger role in the separation mechanism. For native gel electrophoresis, increasing the gel concentration increases resolution as well as increasing the migration times. Another approach involves increasing the gel length, but an increased gel length results in a proportional increase in analysis time (data not shown). According to eq 1, one way to improve resolution, while at the same time reducing the analysis time, is to use a greater electric field strength. To use a greater field strength, the inner diameter of the column was reduced from 2.5 to 1.0 mm. Reducing the gel tube inner diameter, although reducing the maximum amount of protein loadable on the gel, significantly reduced the total analysis time, as shown in Figure 4. Although the analysis was complete in just over 1 h for the 1.0-mm inner diameter column (Figure 4B), an unexpected loss in resolution was seen. Increasing column) to 200 the electric field strength from 40 (2.5" V/cm (1-mm column) should have increased the resolution 7-fold. The experimental loss in resolution may have been due to an increase in Joule heating in the column or deformation of the gel. Although the current was identical for both columns, the current density (current per cross-sectional area) was much greater for the 1-mm column. Second Dimension. The second dimension separation consistsof the switching valve/injector interface and a gradient elution, reversed-phaseHPLC system using a perfusion-type chromatographic column. The main requirement for the second dimension separation is a rapid speed of analysis to provide a sufficiently high sampling of the effluent from the (18) Richards, E. G.; Coll, J. A. Anal. Biochem. 1965, 12, 452-471. (19) Rodbard, D.; Chrambach, A.; Gratzer, W. B. Proc. Nufl. Acud. Sci. U S A . 1970,65,97&977.
Analytical Chemistry, Vol. 66, No. 15, August 1, 1994
2533
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first dimension . The total run time for the second dimension of a comprehensive-type 2D system is determined by the time required to fill the sample loop of the switching valve. For 2D-EP/LC, using a 100-pL loop with a sample elution flow rate of 25 pL/min from the first dimension, a maximum run time of 4 min is allowed for the second dimension HPLC run. In order to accommodate such a short analysis time, high2534 Ana~calChemisiry, Vol. 66, No. 15, August 1, 1994
speed chromatography with perfusive media is employed.16 Such media allow very high flow rates with minimal loss in plate height. For example, a 2.1 X 100 mm column packed with perfusive media can have a flow rate as high as 1.8 mL/ min, its upper limit for a back pressure of 170 bar, without significant loss in resolution. A second requirement for the second dimension is the ability to accommodate the injection
flow rate used in the perfusion chromatographic separation relative to the void volume of the column (230-pL void volume for a 2.1 X 100 mm column), gradient elution can be used as the elution mode of the second dimension. This mode of elution permits more flexibility in terms of the acceptable volumes of injections while at the same time concentrating the sample at the head of the column. To be an effective 2D system, the second dimension must not lose any resolution gained by the first dimension. Loss of resolution is dependent on the sampling volume taken from the effluent of the first dimension. Two or more components resolved in the first dimension separation and sampled as one large volume would be mixed together upon injection into the second dimension. The lower limit of the sampling volume is determined by the speed of the second dimension separation. Figure 5 shows the effect of two different sampling volumes. A samplingvolume of 50 pL (Figure 5A) permits three to five runs or “samplings” across most of the peaks, based on the width of the lines representing one run superimposed on the 3D image. For comparison, a sampling volume of 250 p L (Figure 5B) is limited to approximately one sampling per peak but affords more time to completethe second dimension. The extra time can result in slightly better resolution in the second dimension through the use of a longer column. Although, theoretically the use of a higher samplingfrequency provides the best resolution of the first dimension effluent, from a practical point of view, a high sampling frequency produces fractions beyond the capacity of collection tray (340) unless only a limited portion of the two-dimensional separation is of interest. Furthermore, a higher sampling frequency divides one protein among more fractions, increasing the exposed surface area, which increases the potential for sample loss. Application of 2D-EP/LC. The successful application of 2D-EP/LC to the separation and isolation of complex protein mixtures requires that each dimension have a different or orthogonal separation mechanism. A demonstration of this orthogonality (size/charge vs hydrophobicity) is shown in Figure 3, where, if the 3D image were collapsed along the HPLC dimension, as if only an electrophoretic separation had been performed,lactalbumin, bovine serum albumin, and ovalbumin would be unresolved in the electrophoresis dimension. However, collapsing the 3D image along the electrophoresis dimension results in complete resolution. Had the two separationdimensionsbeen nonorthogonal, samples would have fallen on a diagonal in the 2D separation space such that each dimension would have been highly correlated. Besides being orthogonal, the 2D system must have sufficient sensitivity to detect and collect low levels of proteins. The sensitivity of the 2D-EP/LC system was determined by serial dilutions of a set of standard proteins. From Figure 6, the limit of detection of this system can be estimated to be approximately to 10-20 pmol of protein applied to the gel. The actual limit depends on the protein type and its peak shape after the 2D analysis. Although sensitivity could be greatly increased by fluorescent taggingor using native protein fluorescence, this limit of detection is compatible with the
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lower limit of material that can be analyzed by N-terminal Edman sequencing or mass spectrometry. Fraction collection was verified by running a series of standard proteins, collecting fractions, and assaying the total protein content of each fraction by a Bradford-type assay. After correcting for the delay volume between the UV detector and the fraction collector, a grid image was superimposedon the 2D image to indicate the location of discrete fractions collected. Collecting fractions also permitted the assessment of protein recovery after the 2D separation . Assessment of recovery was made by loading 770 pmol of lactalbumin onto the gel column, collecting the peak into three fractions, and quantifyingthe fractions by amino acid analysis. Total protein recovery from the 2D-EP/LC system for this protein was greater than 90%. The applications most suitable for 2D-EP/LC include those where one or more protein species in a complex mixture of proteins are to be isolated. For example, a crude lysate of a Analytical Chemistry, Vol. 66, No. 15, August 1, 1994
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Tray Column Number (HPLC) Flgure 8. PD-EPILC separation of SF9 cell lysate in which the cell line overexpresses a tyrosine kinase. The grid lines Indicatethe fractions collected for further analysis. Conditions: same as Figure 3 except sampling loops, 500 pL; gradient from 35 to 55% in 9 min with 1-min reequilibration.
cell line that overexpresses a particular protein can be analyzed to isolate the protein of interest. Figure 7 shows the 2DEP/LC separation of the crude cell lysate from an HL60 (human carcinoma) cell line (Figure 7A) and an SF9 (immortalized ovarian insect) cell line (Figure 7B). These figures demonstrate the difficulty often encountered when “real world” samplesare analyzedas compared to test proteins. For example, the streaking of some spots in the electrophoresis dimension appears to be adsorptionof the sampleto gel matrix, Zitex membranes, or connecting tubing. This can cause carryover in the fraction collection process. Furthermore, complex mixtures of diverse components limit the flexibility of optimizing resolution. For example, in Figure 7A, the spot at LC 1 min, EP 35 min limits the changes in the LC gradient to further resolve the components eluting between LC 2 and 3 min. Figure 8 showsthe separationof a cell lysate transfected with baculovirus to overexpress a tyrosine kinase. The grid overlay shows fractions collected for protein sequencing. Although none of the collected fractions yielded an N-terminal protein sequence for positive identification, perhaps due to low expression levels, these analyses clearly demonstrate the resolving power of 2D-EP/LC in the separation and isolation of complex mixtures.
2536 Ana&thIChemistry, Vol. 66, No. 15, August 1, 1994
CONCLUSIONS A micropreparativetwo-dimensional separationsystem has been assembled from commercially available instrumentation, continuous elution, tube gel electrophoresis as the first dimension, and HPLC as the second dimension. The use of orthogonal separation modes (size/charge X hydrophobicity) gives high resolution of complex protein mixtures. Because of the high protein capacity of the gel column (up to 1 nmol), sufficient sample for further analyses can be recovered from the 2D system, even after dilution effects from the HPLC. Automated control of separation system combined with computer imaging of the 2D separation space provides rapid identification of collected fractions. Limitationsof the system includegel columns of limited stability and possible adsorption of samples onto components of the electrophoresis chamber resulting in poor peak shape and sample carryover. ACKNOWLEDGMENT The authors acknowledge Mary Moyer for amino acid analyses of collected fractions. Received for review December 1, 1993. Accepted May 13, 1994.’ Abstract published in Aduunce ACS Abstracts. July 1, 1994.
