Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity. We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity. The resolution of complex samples into components requires sophisticated technology. Most separation techniques are capable of resolving, at most, several dozen components. Cal Giddings (1Giddings J.C. Unified Separation Science. John Wiley & Sons, Inc., New York1991: 126-128Google Scholar) recognized that the combination of two separation techniques is important in the resolution of complex mixtures. If the two separation techniques are based on unrelated characteristics of the sample, then the number of resolution elements is given by the product of the resolution elements of both separation steps. For example, isoelectric focusing and SDS-polyacrylamide gel electrophoresis can, individually, resolve ∼50 components in a protein sample. Their combination, in two-dimensional electrophoresis, can resolve several thousand components (2O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar). Unfortunately, classic two-dimensional electrophoresis requires manual manipulation of the sample. These manipulations, although reasonable for occasional use, are very tedious when performed in large-scale protein analysis projects. Furthermore, detection sensitivity is limited, and relatively large amounts of sample are required to detect weakly expressed components. Column-switching technology is an alternative means of performing two-dimensional separations and is commonly used in chromatographic resolution of complex samples. Most simply, a fraction containing the compound of interest is captured as it elutes from the first column and is transferred to another chromatographic column for additional resolution; fractionation is repeated with different chromatographic methods to achieve the desired purity. In multidimensional chromatography, also known as comprehensive chromatography, a second column is used to sequentially separate all fractions from the first column. These two-dimensional techniques are particularly useful when characterizing extremely complex samples. An early report used two successive chromatographic steps to purify peptides generated from the proteolytic digest of a human immunoglobulin (3Yamamoto H. Manabe T. Okuyama T. Gel permeation chromatography combined with capillary electrophoresis for microanalysis of proteins.J. Chromatogr. 1989; 480: 277-283Google Scholar). Jorgenson and others (4Bushey M.M. Jorgenson J.W. Automated instrumentation for comprehensive two-dimensional high performance liquid chromatography of proteins.Anal. Chem. 1990; 62: 161-167Google Scholar, 5Larmann Jr., J.P. Lemmo A.V. Moore Jr., A.W. Jorgenson J.W. Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis.Electrophoresis. 1993; 14: 439-447Google Scholar, 6Holland L.A. Jorgenson J.W. Separation of nanoliter samples of biological amines by a comprehensive two-dimensional microcolumn liquid chromatography system.Anal. Chem. 1995; 67: 3275-3283Google Scholar, 7Moore Jr., A.W. Jorgenson J.W. Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3448-3455Google Scholar, 8Moore Jr., A.W. Jorgenson J.W. Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3456-3463Google Scholar, 9Opiteck G.J. Lewis K.C. Jorgenson J.W. Anderegg R.J. Comprehensive on-line LC/LC/MS of proteins.Anal. Chem. 1997; 69: 1518-1524Google Scholar, 10Opiteck G.J. Jorgenson J.W. Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.Anal Chem. 1997; 69: 2283-2291Google Scholar, 11Hooker T.F. Jorgenson J.W. A transparent flow-gating interface for the coupling of microcolumn LC with CZE in a comprehensive two-dimensional system.Anal. Chem. 1997; 69: 4134-4142Google Scholar, 12Lemmo A.V. Jorgenson J.W. Transverse flow-gating interface for the coupling of microcolumn-LC with CZE in a comprehensive 2-dimensional system.Anal. Chem. 1993; 65: 1576-1581Google Scholar) have developed elegant multicolumn separations for proteins and peptides. These systems rely on various combinations of size exclusion chromatography, reversed-phase chromatography, and zone electrophoresis to characterize amines, peptides, and proteins. In the most sophisticated version, a mass spectrometer is used to identify components separated by a coupled ion exchange/reversed-phase or size-exclusion/reversed-phase chromatography system (8Moore Jr., A.W. Jorgenson J.W. Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3456-3463Google Scholar, 9Opiteck G.J. Lewis K.C. Jorgenson J.W. Anderegg R.J. Comprehensive on-line LC/LC/MS of proteins.Anal. Chem. 1997; 69: 1518-1524Google Scholar). In the experiments of Jorgenson and co-workers (4Bushey M.M. Jorgenson J.W. Automated instrumentation for comprehensive two-dimensional high performance liquid chromatography of proteins.Anal. Chem. 1990; 62: 161-167Google Scholar, 5Larmann Jr., J.P. Lemmo A.V. Moore Jr., A.W. Jorgenson J.W. Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis.Electrophoresis. 1993; 14: 439-447Google Scholar, 6Holland L.A. Jorgenson J.W. Separation of nanoliter samples of biological amines by a comprehensive two-dimensional microcolumn liquid chromatography system.Anal. Chem. 1995; 67: 3275-3283Google Scholar, 7Moore Jr., A.W. Jorgenson J.W. Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3448-3455Google Scholar, 8Moore Jr., A.W. Jorgenson J.W. Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3456-3463Google Scholar, 9Opiteck G.J. Lewis K.C. Jorgenson J.W. Anderegg R.J. Comprehensive on-line LC/LC/MS of proteins.Anal. Chem. 1997; 69: 1518-1524Google Scholar, 10Opiteck G.J. Jorgenson J.W. Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.Anal Chem. 1997; 69: 2283-2291Google Scholar, 11Hooker T.F. Jorgenson J.W. A transparent flow-gating interface for the coupling of microcolumn LC with CZE in a comprehensive two-dimensional system.Anal. Chem. 1997; 69: 4134-4142Google Scholar, 12Lemmo A.V. Jorgenson J.W. Transverse flow-gating interface for the coupling of microcolumn-LC with CZE in a comprehensive 2-dimensional system.Anal. Chem. 1993; 65: 1576-1581Google Scholar) (Fig. 1) a portion of each peak that elutes from a microbore chromatography column is sequentially injected into an electrophoresis capillary, where overlapping components are resolved. Because electrophoresis is faster than the elution time of a chromatographic peak, a portion of each peak from the chromatography column is sampled by the electrophoresis capillary. A two-dimensional separation is reconstructed by plotting the electrophoresis separations next to each other. The appearance of the plot is quite similar to a classic two-dimensional electropherogram, although it is generated by combining liquid chromatography with capillary electrophoresis. Sequential separation offers two advantages over conventional two-dimensional electrophoresis, where components are separated simultaneously. First, because components are detected sequentially, a sensitive detector can be incorporated into the instrument. Second, the separation is automated; once the sample is injected, there is no further operator intervention. However, sequential separation is usually slower than parallel separation. The interface between the two columns is key to the performance of the system. Jorgenson and co-workers (11Hooker T.F. Jorgenson J.W. A transparent flow-gating interface for the coupling of microcolumn LC with CZE in a comprehensive two-dimensional system.Anal. Chem. 1997; 69: 4134-4142Google Scholar, 12Lemmo A.V. Jorgenson J.W. Transverse flow-gating interface for the coupling of microcolumn-LC with CZE in a comprehensive 2-dimensional system.Anal. Chem. 1993; 65: 1576-1581Google Scholar) have demonstrated an elegant flow-gated interface to couple an HPLC 1The abbreviations used are: HPLC, high performance liquid chromatography; FQ, 3-(2-furoyl)quinoline-2-carboxaldehyde; DALT, dalton; CAPS, 3-(cyclohexylamino)propanesulfonic acid. 1The abbreviations used are: HPLC, high performance liquid chromatography; FQ, 3-(2-furoyl)quinoline-2-carboxaldehyde; DALT, dalton; CAPS, 3-(cyclohexylamino)propanesulfonic acid. column with a capillary electrophoresis column. This interface uses a flow of buffer to control injections of chromatographic fractions into a capillary zone electrophoresis column. The effluent continually migrates from the HPLC capillary and is swept to waste by the cross-flow in the interface. To inject a fraction into the electrophoresis capillary, the cross-flow of buffer is halted, and a plug of HPLC effluent forms in the interface. Potential is applied from the buffer vial across the electrophoresis capillary to the detection end of the capillary, which injects the HPLC effluent into the capillary. The buffer flow is reapplied to the interface, terminating the injection. The electric field remains applied across the capillary to separate the injected components. Unfortunately, to avoid continuous injection of the HPLC components during the electrophoresis step, only a small portion of each peak is injected into the electrophoresis column; most of the HPLC effluent is directed to waste. We have reported the use of capillary electrophoresis for the ultrasensitive analysis of proteins (13Pinto D.M. Arriaga E.A. Craig D. Angelova J. Sharma N. Ahmadzadeh H. Dovichi N.J. Boulet C.A. Picomolar assay of native proteins by capillary electrophoresis, precolumn labeling, submicellar separation, and laser-induced fluorescence detection.Anal. Chem. 1997; 69: 3015-3021Google Scholar, 14Lee I.H. Pinto D. Arriaga E.A. Zhang Z. Dovichi N.J. Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.Anal. Chem. 1998; 70: 4546-4548Google Scholar). Our systems rely on the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) to convert lysine residues to highly fluorescent product. This reagent is non-fluorescent until it reacts with a primary amine in the presence of a nucleophile. The use of a fluorogenic reagent reduces the background signal significantly compared with the use of conventional fluorescent reagents. When coupled with a high sensitivity laser-induced fluorescence detector, detection limits of a few zeptomoles are routinely achieved. We have more recently demonstrated the separation of proteins from a single cancer cell using both submicellar and SDS-DALT electrophoresis, where the latter term refers to size-based electrophoresis of proteins in a sieving matrix (15Zhang Z. Krylov S. Arriaga E.A. Polakowski R. Dovichi N.J. One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell.Anal. Chem. 2000; 72: 318-322Google Scholar, 16Hu S. Zhang L. Cook L.M. Dovichi N.J. Capillary SDS-DALT electrophoresis of proteins in a single human cancer cell.Electrophoresis. 2001; 22: 3677-3682Google Scholar). Unfortunately, our one-dimensional separations are unable to resolve the large number of components present in a complex cellular lysate. In this paper, we report the development of a fully automated, two-dimensional electrophoresis system to further resolve these components. Our system differs from that of Jorgenson and co-workers (4Bushey M.M. Jorgenson J.W. Automated instrumentation for comprehensive two-dimensional high performance liquid chromatography of proteins.Anal. Chem. 1990; 62: 161-167Google Scholar, 5Larmann Jr., J.P. Lemmo A.V. Moore Jr., A.W. Jorgenson J.W. Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis.Electrophoresis. 1993; 14: 439-447Google Scholar, 6Holland L.A. Jorgenson J.W. Separation of nanoliter samples of biological amines by a comprehensive two-dimensional microcolumn liquid chromatography system.Anal. Chem. 1995; 67: 3275-3283Google Scholar, 7Moore Jr., A.W. Jorgenson J.W. Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3448-3455Google Scholar, 8Moore Jr., A.W. Jorgenson J.W. Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.Anal. Chem. 1995; 67: 3456-3463Google Scholar, 9Opiteck G.J. Lewis K.C. Jorgenson J.W. Anderegg R.J. Comprehensive on-line LC/LC/MS of proteins.Anal. Chem. 1997; 69: 1518-1524Google Scholar, 10Opiteck G.J. Jorgenson J.W. Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.Anal Chem. 1997; 69: 2283-2291Google Scholar, 11Hooker T.F. Jorgenson J.W. A transparent flow-gating interface for the coupling of microcolumn LC with CZE in a comprehensive two-dimensional system.Anal. Chem. 1997; 69: 4134-4142Google Scholar, 12Lemmo A.V. Jorgenson J.W. Transverse flow-gating interface for the coupling of microcolumn-LC with CZE in a comprehensive 2-dimensional system.Anal. Chem. 1993; 65: 1576-1581Google Scholar) in three important respects. First, we use capillary electrophoresis in both dimensions of the separation, which eliminates the use of chromatography, with its associated pumps, valves, and large sample volume. Second, we transfer the entire fraction from the first capillary to the second, eliminating the loss of sample in the flow-gated interface. Third, we use an extremely high sensitivity fluorescence detector, which allows us to monitor zeptomoles of proteins. The labeling reagent FQ and cyanide are from Molecular Probes. Water is deionized and distilled (Barnstead). All other reagents are from Sigma. Protein extracts are prepared from HT29 human adenocarcinoma cells (14Lee I.H. Pinto D. Arriaga E.A. Zhang Z. Dovichi N.J. Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.Anal. Chem. 1998; 70: 4546-4548Google Scholar). Roughly 106 cells are lysed by sonication and labeled with 100 nmol of FQ in the presence of 2.5 mm potassium cyanide at 65 °C for 5 min. The FQ-labeled cell extract is diluted 50-fold in 10 mm phosphate buffer, pH 7, and stored on ice (15Zhang Z. Krylov S. Arriaga E.A. Polakowski R. Dovichi N.J. One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell.Anal. Chem. 2000; 72: 318-322Google Scholar). A 10 mm HEPES and 5 mm SDS buffer, pH 7.5, is used for the first dimension separation whereas the second dimension separation buffer is 40 mm CAPS and 5 mm SDS, pH 11.1. Fig. 2 presents a schematic diagram of the fully automated two-dimensional capillary electrophoresis instrument. Two-dimensional electrophoresis is driven by two 0–30,000-V power supplies (Ultra Volt) that are controlled by a Macintosh II computer and driven by software written in Labview (National Instruments). Separation is performed in two 40-cm-long, 50-μm-inner diameter, 138-μm-outer diameter polyamide-coated fused silica capillaries (Polymicro). The polyamide coating is burned away from the detector end with a gentle flame. Buffer 2 (CAPS) inlet and outlet capillaries have 145-μm inner diameters/367-μm outer diameters with lengths of 6 and 15 cm. The running Buffer 1 (HEPES), running Buffer 2, and detector buffer reservoirs are kept at equal heights to prevent siphoning through the capillary during the experiment. The black square in Fig. 2 represents a modified version of Jorgenson's interface that aligns the two separating capillaries and the two waste capillaries. The machine shop at the Chemistry Department, University of Alberta drilled two 370-μm diameter holes in a piece of clear Lexan fitted in a cross (Valco). Chamfered holes were also drilled to hold ferrule fittings. Before assembly, the interface is rinsed with distilled water to remove any particles and air trapped in the cross. With the aid of a microscope, each of the four capillaries are threaded through 1/16-inch sleeves and carefully lined in position. The capillaries are held in place by tightening the ferrules in the Valco cross. The first dimension capillary inlet is placed in the injection buffer reservoir whereas the second dimension capillary outlet is connected into the sheath-flow cuvette. The 6-cm-long Buffer 2 outlet capillary is placed in the Buffer 2 reservoir whereas the 15-cm-long Buffer 2 inlet capillary is connected to a wash-bottle located above the interface to allow gravity flow. Flow in the Buffer 2 inlet is controlled using a low pressure shut-off valve (Upchurch). Prior to a separation, the capillaries are washed by purging with 0.25 m NaOH for 5 min with nitrogen gas. To ensure flow through both capillaries, the Buffer 2 inlet valve remains closed, and the Buffer 2 outlet capillary is plugged. The second dimension capillary is filled with running Buffer 2 by whereas both the Buffer 2 inlet and outlet are plugged. The first dimension capillary is with Buffer 1 whereas the Buffer 2 inlet valve is and the Buffer 2 outlet capillary is injection is by a of to the sheath-flow the valve is and the waste capillary outlet is Arriaga E.A. Zhang Z. M.M. Dovichi N.J. for Chem. 2000; 72: Scholar). a separation in the first dimension fractions are transferred to the second dimension capillary. 10 is applied to power and is applied to power 2 for min the waste valve remains the separation, the waste valve is closed, and second dimension separation A fraction from the first dimension is into the second dimension capillary by with power 1 and 10 with power 2 for Once the fraction is the second dimension separation is performed by 10 to power 1 is to ensure the is applied to the inlet and outlet of capillary which flow of sample in the first capillary during the second dimension separation. The of fraction transfer and second dimension separation are repeated computer fluorescence detection with a sheath-flow is used to monitor the separated labeled proteins Dovichi N.J. analysis by capillary zone electrophoresis and laser-induced Scholar, S. Dovichi N.J. High sensitivity fluorescence detector for of separated by capillary zone Chromatogr. 1989; 480: Scholar). The sample from the second dimension capillary is in the sheath-flow and with an with a To each separation, the two-dimensional electrophoresis first to in a one-dimensional capillaries were filled with the The injection end of the capillary held at the interface at and the detector at In this the sample is separated at across an capillary. Fig. presents two capillary generated from of the HT29 The separation performed in a pH 7.5 buffer, and the dimension performed in a pH 11.1 Roughly two dozen components are in the low pH separation whereas four dozen components are in the high pH separation. The between and which that of the sample across the interface The then to in a two-dimensional were to electrophoresis in the first capillary at pH 7.5, and a fraction transferred for further separation at pH 11.1. This successive transfer and separation of fractions repeated 100 Fig. presents a two-dimensional generated with the sequential of these two electrophoresis The by the each column of the two-dimensional and the the sample separated with the second dimension capillary. The by the each of the two-dimensional and to the from a separation generated by the first dimension capillary. Fig. 5 is an of the between 1 and fractions from the first capillary and in the second Fig. 6 is a of the plot of the of Fig. is an important in capillary separations of labeled proteins. The most labeling reagents the of lysine has to all lysine and a of is If there are lysine there are 1 K.C. J. Zhang Dovichi N.J. of a single fluorescent to peptides for by capillary zone Chromatogr. Scholar). For example, has lysine there are fluorescent from the labeling of these can have a different to a complex with separation We have that the use of FQ as a labeling reagent and the use of an at submicellar very high resolution of the labeling into a peak with as high as (14Lee I.H. Pinto D. Arriaga E.A. Zhang Z. Dovichi N.J. Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.Anal. Chem. 1998; 70: 4546-4548Google Scholar). The FQ reagent reacts with primary amines to a product. We that the ion with lysine also a that the on the protein is of the of is to use of high SDS to proteins at a to a and a very separation in the of a sieving during electrophoresis. Protein on the inner of the separation capillary remains a We have demonstrated that proteins to the capillary in and are to in the analysis of protein extracts Arriaga E.A. Zhang Z. M.M. Dovichi N.J. for Chem. 2000; 72: Scholar). In this we the capillary between to remove proteins that with We have demonstrated that modified are useful in protein during capillary electrophoresis R. Ahmadzadeh H. Dovichi N.J. based on and and a of for capillary electrophoresis.Electrophoresis. 1998; Scholar). be required for the analysis of highly and highly which were during our is important that the two separation are components that are in the first dimension are separated in the We the separation of protein performed in electrophoresis at pH from to In all separation with separation of there were in with pH and the separation quite from pH to The significantly at pH because of the of and We the pH 7.5 and 11.1 for this because the two quite different separation for the cellular sample. We are to perform SDS-DALT electrophoresis in the first dimension and submicellar electrophoresis in the second which two-dimensional separation. of the two-dimensional can be to the characteristics of the one-dimensional separations used to the time to the two-dimensional electropherogram. In Fig. the first from the pH 7.5 capillary at min. This generated with the as a one-dimensional electrophoresis with an capillary min for the first to through the first capillary and to the interface. When the two-dimensional electropherogram, we performed a separation of the sample by an electric field to the first dimension capillary for min. Potential be applied to the first capillary for an additional 5 min to the separation. This is only applied during the when fractions are from the first capillary and transferred to the The transfer is 6 and are required to all fractions from the first capillary and transfer to the Roughly min are required to separate the sample in the pH 11.1 buffer, using the capillary in Fig. min for the to the detector, and the separation another min until the migrates from the capillary. it 6 min for the to through the second dimension capillary, and the separation is also 6 min. To the analysis we inject fractions into the second capillary at a two fractions separation in the second dimension capillary. The transfer are that the of the first fraction with the of the second The analysis time is are required to the two-dimensional of Fig. However, this separation is performed operator intervention or We have demonstrated that a capillary sheath-flow is an detector in high Lewis J. R. H. Ahmadzadeh H. Dovichi N.J. A electrophoresis system for and Scholar, Lewis Zhang Dovichi N.J. Capillary based on a sheath-flow 2000; Scholar, J. Cook L.M. Dovichi N.J. Two-dimensional fluorescence for by capillary electrophoresis.Anal. Chem. 2001; Scholar). We to the two-dimensional electrophoresis that samples can be analyzed in A single samples with no operator intervention. The with exquisite sensitivity. Our protein sample the extract from 106 HT29 cancer which were prepared in a volume. The sample labeled and diluted 50-fold analysis. For an of labeled proteins injected into the capillary, and the of protein injected is to that in a single The detection of the is a few zeptomoles of labeled and the components in Fig. 5 are present at a few thousand is important to of this system. The fluorescence detector, although providing exquisite very on the of the An alternative system a mass spectrometer as a the sensitivity of the fluorescence detector, the mass spectrometer to identify the proteins.

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