Rapid Determination of Protein Molecular Weight by the Ferguson Method and Multiplexed Capillary Electrophoresis Yan He and Edward S. Yeung* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 Received January 30, 2002
Abstract: A method based on the use of the Ferguson method and multiplexed sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) with UV detection was demonstrated for the rapid determination of molecular weights of proteins. The method employs a capillary array where different uncoated capillaries are filled with gel buffers containing different concentrations of poly(ethylene oxide) (PEO). All data required to construct Ferguson plots and universal calibration curves for the determination of the molecular weights of diverse types of proteins are generated simultaneously in an eightcapillary array within 20 min. Keywords: electrophoresis • SDS-PAGE • size separation
1. Introduction With the imminent completion of the Human Genome Project, scientists are beginning to decipher the structure and function of the numerous proteins translated from the genetic sequence, creating the field which is dubbed proteomics.1,2 Proteomics seeks to characterize the entire complement of proteins expressed in a cell under defined conditions. Although the human genome may contain as few as 30 000 genes, many more proteins are produced through post-translational modifications such as proteolytic processing, glycosylation, and phosphorylation. Consequently, there is a critical need for analytical techniques that can provide high-throughput characterization of the numerous and diversified proteins.3 Determination of molecular weight is one of the most important aspects of protein characterization. Mass spectrometry (MS) with either matrix-assisted laser desorption and ionization4 (MALDI) or electrospray ionization5 (ESI) is a natural choice for accurate molecular weight determinations. While several proteins can be sized in the same sample, throughput is still limited by either sequential sample introduction or critically regimented sample preparation. Ideally, one wants molecular weight determinations to be performed directly on complex biological matrixes. Furthermore, fragmentation during ionization or inclusion of solvent molecules can lead to incorrect sizing in mass spectrometry. Finally, variations in ionization efficiency imply that accurate quantitative results are difficult to obtain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been one of the most commonly used methods for the determination of protein molecular weight for 10.1021/pr025507i CCC: $22.00
2002 American Chemical Society
over three decades.6 In particular, the combination of isoelectric focusing in the 2D-gel format has made SDS-PAGE a key tool for proteomics research. In SDS-PAGE, proteins are separated by size on cross-linked polyacrylamide slab gels with low applied electric fields (10-30 V/cm) and then typically detected by post-separation staining. SDS-PAGE has the advantage of simultaneously analyzing multiple samples on one gel. However, the process is slow and labor intensive due to the requirement of manual preparation of the gel, low separation voltage, and post-separation staining of the proteins. In an effort to overcome some of these problems, Cohen et al.7 examined the sizing of proteins in capillaries by SDS-PAGE. Migration in capillary gel electrophoresis (CGE) was much more rapid than SDS-PAGE in the slab format due to the use of high electric fields (200-600 V/cm). Separation by SDS-CGE is typically achieved in less than 20 min with on-column absorbance detection. It is even possible to incorporate automation.8-11 Nevertheless, SDS-CGE performed with conventional CE instruments, where only a single capillary is used, is hampered by the low throughput. The problem becomes more serious when the analysis of diversified proteins by several complementary methods is required. Size determinations of proteins by SDS-PAGE and SDSCGE are based on the same assumption that most proteins bind SDS at the ratio 1:1.412,13 and thus have identical charge-tomass ratios. However, many protein-SDS complexes do not possess the standard charge-to-mass ratio because they bind SDS differentially. These proteins include highly basic proteins, glycoproteins and lipoproteins. For these proteins, standard calibration curves based on conventional SDS-CGE using a single gel concentration would give substantially higher or lower estimates of the molecular weight.14,15 Therefore, the Ferguson method16 should be used to account for the anomalous behavior of various proteins to provide more accurate estimates of the molecular weights. However, the Ferguson method requires the analysis of each protein under several different gel concentrations. This makes the use of traditional slab gel protocols even more time-consuming and labor intensive. Ferguson analysis of proteins by single capillary SDS-CGE can be automated to reduce the labor cost.10,11 The availability of multiple-capillary electrophoresis instrumentation17,18 provides for simultaneous measurements under multiple separation conditions. Therefore, to further improve the throughput for protein sizing, the use of the Ferguson method in conjunction with multiplexed SDS-CGE is investigated in this work. Rugged operation is made possible by the use of uncoated capillary columns. Journal of Proteome Research 2002, 1, 273-277
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Rapid Determination of Protein Molecular Weight
technical notes
Figure 1. Separation of protein mixture containing seven standard proteins by PEO gel. Conditions: capillary, 50 cm × 75 mm i.d., effective length, 30 cm; gel buffer, TRIS, 0.1 M, CHES, 0.1 M, SDS, 0.1%, PEO (MW 600 000), 1.5%, pH 8.7; applied voltage, 12 kV; injection, -10 kV × 20 s. Peaks: 1 ) R-lactalbumin (MW 14 200); 2 ) carbonic anhydrase (MW 29 000); 3 ) ovalbumin (MW 45 000); 4 ) bovine serum albumin (MW 66 000); 5 ) phosphorylase b (MW 97 400); 6 ) β-galactosidase (MW 116 000); 7 ) myosin (MW 205 000). OG, orange G used as tracking dye.
2. Experimental Section 2.1. Apparatus. All SDS-CGE separations of proteins were optimized on a commercial single-capillary CE instrument (ISCO, Lincoln, NE, model 3140 Electropherograph System) before employing multidimensional multiplexed CE. Bare fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with 30-cm effective length and 55-cm total length (75-mm i.d. and 360-mm o.d.) were used. A negative power supply was used to drive the electrophoretic separation. The experimental CE setup for eight-capillary array electrophoresis is similar to the 96-capillary array system described in ref 17. Here, the capillaries were packed side by side at the detection window and clamped between two flat surfaces of a plastic mount. At the outlet end, capillaries 1 and 2 were immersed in gel buffer 1 (G1) containing 0.75% poly(ethylene oxide) (PEO), capillaries 3 and 4 in gel buffer 2 (G2) containing 1.10% PEO, capillaries 5 and 6 in gel buffer 3 (G3) containing 1.3% PEO, and capillaries 7 and 8 in gel buffer 4 (G4) containing 1.5% PEO. A fourposition valve was used to distribute nitrogen gas from a gas cylinder to eight gel buffer vials to simultaneously fill eight capillaries with four gel buffers. At the injection end, which is grounded, the eight-capillary array was spread and mounted on a copper plate with dimensions that fit into one row of a 96-well microtiter plate for sample introduction. Eight goldcoated pins (Mill-Max Mfg. Corp., Oyster Bay, NY) were mounted on the copper plate near each capillary tip to serve as individual electrodes, with the capillary tips slightly extended (∼0.5 mm) beyond the electrodes to guarantee contact with small-volume samples. A negative high-voltage power supply (Glassman High Voltage Inc., Whitehorse Station, NJ) was used to drive the electrophoresis. The light source, interference filter, capillary-array holder, and photodiode array (PDA) detector were all contained in a light-tight metal box attached to an optical table. A 213.9-nm zinc lamp (model ZN-2138, Cole-Parmer, Vernon Hills, IL) was used as the light source for UV absorption detection. The transmitted light from the capillary array passed through an interference filter (Oriel, Stratford, CT) and a quartz lens (Nikon; focal length ) 105 mm; f/4.5). An inverted image of the capillary 274
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array at a nominal magnification factor of 1.2 was created by the quartz lens on the face of the PDA. The PDA (Hamamatsu model S5964, Hamamatsu, Japan) incorporated a linear image sensor chip (1024 diodes, 25 mm in width, 2.5 mm in height), a driver/amplifier circuit, and a temperature controller. The built-in driver/amplifier circuit was interfaced to an IBMcompatible computer via a National Instruments PCI E Series multifunction 16-bit data-acquisition board. All codes used to operate the PDA and to acquire the data were written in-house using Labview 5.0 software (National Instruments, Austin, TX). The raw data sets were converted into single-channel electropherograms by another in-house Labview program. Data treatment and analysis were performed using Microsoft Excel 97 and Grams/32 5.05 (Galactic Industries, Salem, NH). 2.2. Chemicals. PEO (MW 100 000) and PEO (MW 600 000) were purchased from Aldrich (Milwaukee, WI). 2-Mercaptoethanol, the protein standard test mixture, and SDS-containing wash solution were purchased from Beckman-Coulter (Fullerton, CA). The CHES buffer, β-lactoglobulin A, β-casein, lactoperoxidase, sample dilution buffer, and orange G (OG) were purchased from Sigma (St. Louis, MO). Trisma-base [tris(hydroxymethyl)aminomethane] (TRIS) and sodium dodecyl sulfate were obtained from Fisher Scientific (Fairlawn, NJ). The water used to prepare buffer and reaction solutions was purified by a Milli-Q purification system (Millipore, Worcester, MA). 2.3. Procedures. The separation buffer (TRIS, 0.1 M; CHES, 0.1 M; SDS, 0.1%; pH 8.7) was first prepared. The stock gel buffer containing 1.5% PEO (MW 600 000) was prepared by dissolving PEO powder in the separation buffer and stirring at a high speed for 4-6 h. This gel buffer, named G4, was then vacuum degassed and left undisturbed for 1 day to make it more homogeneous before use. G4 was diluted by the separation buffer to 86, 72, and 50% of the original concentration to prepare the diluted gel buffers G3, G2, and G1. The diluted gel buffers were vortexed for 5 min and left undisturbed for 1 day prior to use. The protein standard mixture and the proteins of interest were diluted to 0.1-1 mg/mL and heated to 95 °C for 10 min
technical notes
Figure 2. Standard calibration curve of logarithm molecular weight (log MW) vs reciprocal of relative migration time (1/RMT). Relative migration time is the migration time of a protein divided by that of OG.
after addition of 2% 2-mercaptoethanol and 1% (v/v) orange G. The samples were then cooled on ice for 3 min before injection. 2-Mercaptoethanol was used as a reducing agent and orange G as the internal standard. The eight-capillary array was washed with SDS-containing wash solution for 7 min, water for 5 min, and filled with the different gel buffers (G1, G2, G3, and G4) for 8 min. The pretreated protein standards and samples were separately injected at -10 kV for 20 s into the 8 capillaries. After injection, the inlets of the capillaries were immersed in the gel buffers, and -12 kV was applied to start the 20-min separation.
3. Results and Discussion 3.1. Size Separation of Proteins in Uncoated Capillary and Replaceable PEO Gel. In previous works on the determination of molecular weight by SDS-CGE, a coated capillary was usually deployed to eliminate electroosmotic flow (EOF) and to minimize nonspecific adsorption of proteins on the inner surface of the capillary.7,10,11 The use of coated capillaries in multiplexed CE is, however, not practical due to the high cost and reduced ruggedness compared to that of uncoated capillaries. So, the possibility of using uncoated capillaries was investigated in this study.
He and Yeung
The procedure described in ref 9 was first followed to prepare the gel buffer containing 3% PEO (MW 100 000). It was found that dissolution of the polymer in the buffer was very slow. Homogeneous and clear solutions were not obtained even after violent stirring for 12 h. Then PEO with higher molecular weight (MW 600 000) was tested. PEO (1.5%) (MW 600 000) can be dissolved into the buffer quickly, and a homogeneous and transparent gel buffer was obtained after several hours of stirring. In addition, the viscosity of 1.5% PEO (MW 600 000) is similar to that of the gel based on 3% PEO (MW 100 000). Using the former gel buffer, the separation of a standard protein test mixture was evaluated (Figure 1). As can be seen, good separation of seven proteins was achieved within 20 min, which is comparable to the separation using a coated capillary.10,11 In addition, six of the seven peaks were sharp and symmetric while the peak of BSA was only slightly broadened. Hence, the adsorption of SDS-protein complexes on the capillary wall should not be a problem for most proteins. The reason for the broadening of the BSA peak may be 2-fold. One possibility is that the capillary inner wall is still incompletely coated by the PEO. The other possibility is that the SDS binding on the protein is not uniform, thereby exposing hydrophobic pockets that favor adsorption. Figure 2 shows a good linear relationship between the logarithm of MW and the reciprocal of the relative migration time for seven proteins. The result suggests that the influence of any residual EOF on the linear relationship is negligible in this protocol. While protein standards show a linear relationship in the calibration plot, there are some proteins that behave differently and do not fall on the standard regression line. This unusual behavior can be clearly seen in Figure 3 in the separation of glycoproteins and nonglycosylated proteins. β-Lactoglobulin A (MW 18 000) is a nonglycosylated protein, and its migration behavior is normal, with migration time between those of R-lactalbumin (MW 14 200) and carbonic anhydrase (MW 29 000). β-Casein and lactoperoxidase are glycoproteins, and their migration behaviors are anomalous as compared to the protein standards. For example, the migration time of β-casein (MW 25 000) is even longer than that of ovalbumin although it is much smaller than ovalbumin (MW 45 000). Simply using the calibration curve shown in Figure 2 gives a molecular weight that is off by 82%. The increased migration time is caused by the lower binding ratio of SDS due
Figure 3. Size separation of a nonconjugated protein and glycoproteins by SDS-CGE. Peaks: 1 ) β-lactoglobulin A (MW 18 000), 2 ) β-casein (MW 25 000), 3 ) lactoperoxidase (MW 76 000). Other conditions are the same as those in Figure 1. Journal of Proteome Research • Vol. 1, No. 3, 2002 275
Rapid Determination of Protein Molecular Weight
technical notes
Figure 4. Separation of protein standards and glycoproteins by multiplexed SDS-CGE. Conditions: see the Experimental Section. Gn-S and Gn-PS are protein samples (S) or protein standards (PS) separated by Gn gel buffer.
to the presence of carbohydrate side chains in glycoproteins. Therefore, the Ferguson method should be used to achieve a more precise estimate of the molecular weight of these types of molecules. 3.2. Separation of Proteins by Multiplexed SDS-CGE. The Ferguson method is based on two plots.16 First, the Ferguson plots are constructed by plotting the logarithm of the reciprocal relative migration time of the individual proteins as a function of varying gel concentrations. The negative of the slopes of these lines are called the retardation coefficients (Kr). Second, a universal MW calibration curve can be drawn by plotting the logarithms of molecular weights as a function of the square roots of these retardation coefficients. The calibration curve is applicable to not only nonconjugated proteins but also differently conjugated proteins, such as glycoproteins and lipoproteins. By using an eight-capillary array, separations of protein standards and proteins of interest at four different concentrations of gel buffers were recorded simultaneously (Figure 4). In addition, cross-contamination from using different gel concentrations in the same capillary one after another was also eliminated. The variation of migration times among different capillaries is an important concern when using multiplexed SDS-CGE.18 In practice, much care was taken to ensure that each capillary is cut with the same total and effective lengths in order to reduce the variability. The tracking dye OG was further used to measure and to correct for the variation of migration times among different capillaries filled with the same or with different concentrations of PEO. It was found that the variation in migration times between capillaries filled with the same gel was in the range of 0.4-1.8%, which is comparable to the runto-run variation using a single capillary. More interestingly, the variation in migration times among capillaries filled with different concentrations of gels was less than 4.0%. This result suggests that the EOF remains low and relatively constant within this range of PEO gel concentration. The electropherograms in Figure 4 were transformed into the Ferguson plots displayed in Figure 5. A universal calibration curve (solid line) based on the data from Figure 5a was constructed by plotting the logarithm of molecular weight versus the square root of retardation coefficient of the protein 276
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Figure 5. Ferguson plots of (a) protein standards, bottom plot; and (b) test proteins, top plot. Conditions are the same as those in Figure 4.
standards (Figure 6). The curve was then used to determine the molecular weights of three proteins of interest based on their retardation coefficients obtained from Figure 5b. Table 1 compares the molecular weights of proteins determined from a standard calibration curve and from the Ferguson calibration curve. It is clear that the Ferguson method gives more precise
technical notes
He and Yeung
the 2 days required by the use of single capillary SDS-CGE. The use of absorption at 214 nm provides universal detection and quantitation of all proteins. Indeed, because absorption primarily is due to the amide bonds, quantitative determinations are expected to be superior to traditional protein staining protocols in slab gels. Compared to commercial SDS-PAGE instrumentation such as the Pharmacia Phast-system, the present approach is faster, less labor intensive as a result of automation, and does not require an additional step for protein visualization. Compared to MS measurements, the absence of extensive sample workup is an advantage here.
Acknowledgment. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa Sate University under Contract No. W-7405-Eng-82. This work was supported by the Director of Science, Office of Biological and Environmental Research, and the National Institutes of Health. Figure 6. Universal Ferguson calibration curve of logarithm molecular weight (log MW) vs square root of the retardation coefficient (Sqrt Kr). Table 1. Comparison of Estimated Molecular Weight of Proteins Using Standard Calibration Curve and Ferguson Universal Calibration Curve
References (1) (2) (3) (4) (5) (6) (7)
molecular wt proteins
lit.19,20 (k)
standard (k)
Ferguson (k)
β-lactoglobulin A β-casein lactoperoxidase
18 25 76
14 (22%) 45 (80%) 120 (58%)
20 (11%) 14 (38%) 65 (14%)
MW estimation for both nonconjugated proteins (β-lactoglobulin A) and glycoproteins (β-casein and lactoperoxidase). The improvement in precision was more significant for glycoproteins. It should be noted that the MW deviation from the expected value of β-casein is still significant even when the Ferguson method is employed. This deviation can be attributed to the carbohydrate moiety in β-casein, creating a significant change in the conformation of the SDS-protein complex and causing a nonlinear variation of size with molecular weight. In summary, we have demonstrated a high-throughput method for protein sizing that requires minimal sample workup. With the use of a 96-capillary array, up to 23 proteins can be analyzed within roughly 0.5 h, which is much faster than
(8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
Williams, K. L. Electrophoresis 1999, 20, 678-688. Quadroni, M.; James, P. Electrophoresis 1999, 20, 664-677. Wehr, T. LC-GC 2001, 19, 702-711. Ryzhov, V.; Fenselau, C. Anal. Chem. 2001, 73, 746-750. Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 2488-2499. Weber, K.; Osborn, M. J. Biol. Chem. 1969, 244, 4406-4412. Cohen, A. S.; Paulus, A.; Karger, B. L. J. Chromatogr. 1987, 397, 409-417. Wu, D.; Regnier, F. E. J. Chromatogr. 1992, 608, 349-356. Kalman, B.; Guttman, A. J. Chromatogr. A 1994, 680, 375-381. Guttman, A.; Shieh, P.; Lindahl, J.; Cooke, N. J. Chromatogr. A 1994, 676, 227-231. Werner, W. E.; Demorest, D. M.; Wiktorowicz, J. E. Electrophoresis 1993, 14, 759-763. Reynolds, J. A.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002-1007. Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161-5165. Chrambach, A. The Practice of Quantitative Gel Electrophoresis; VCH: Deerfield Beach, FL, 1985. Andrews, A. T. Electrophoresis, 2nd ed.; Clarendon Press: Oxford, 1986. Ferguson, K. A. Metab. Clin. Exp. 1964, 13, 985-1002. Gong, X.; Yeung, E. S. Anal. Chem. 1999, 71, 4989-4996. Xue, G.; Pang, H.-M.; Yeung, E. S. Anal. Chem. 1999, 71, 26422649. Atlas of Protein and Genomic Sequences, CD-ROM produced by National Biomedical Research Foundation, June 30, 1992. Durchschlag, H.; Christl, P.; Jaenicke, R. Prog. Colloid Polym. Sci. 1991, 86, 41-56.
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