Accuracy in the Determination of Isoelectric Points ... - ACS Publications

To evaluate the accuracy of isoelectric point determination by capillary isoelectric focusing, the pI values of nine proteins and a peptide, the pI va...
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Anal. Chem. 2000, 72, 4747-4757

Accuracy in the Determination of Isoelectric Points of Some Proteins and a Peptide by Capillary Isoelectric Focusing: Utility of Synthetic Peptides as Isoelectric Point Markers Kiyohito Shimura,*,† Wang Zhi,†,‡ Hiroyuki Matsumoto,§ and Ken-ichi Kasai†

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan, and Laboratory of Molecular BioPhotonics, 5000 Hirakuchi, Hamakita, Shizuoka 434, Japan

To evaluate the accuracy of isoelectric point determination by capillary isoelectric focusing, the pI values of nine proteins and a peptide, the pI values of which had been determined by other methods and ranging pI 3.55-9.60, were determined by capillary isoelectric focusing by cofocusing of recently developed peptide pI markers ranging 3.38-10.17, and the consistency of the pI values was examined. Isoelectric focusing was carried out in neutral polymer-coated capillaries, and the pH gradient was mobilized by pressure toward the cathode, to detect samples with absorption at 280 nm at a fixed detection point. Carrier ampholytes from two different suppliers and in different pH ranges were used. The sharp peaks of the highly pure peptide pI markers greatly facilitated the unambiguous identification of the peaks. When a carrier ampholyte ranging over the acidic side was used, the detection of acidic pI samples was anomalously delayed. This could be partly mitigated by reducing the viscosity of the anode solution in comparison with the pH gradient formed in the capillary. Since the detection times vs the pH relationships were not linear in most cases, the use of a linear calibration line over an entire pH gradient would be erroneous. Instead, the pI values of samples were calculated by assuming a linear relation for pH against detection time between two flanking marker peptides. Close agreement between the pI values, determined by capillary isoelectric focusing, and the reference values of the samples was observed within an average difference range of 0.04-0.08 pH unit with a sample consumption of 10-100 ng within 30-60 min. Some carrier ampholytes were preferentially more effective at either the acidic or the basic side of the pH gradient. For confirmation of the completion of focusing, the use of two different focusing times is recommended. The isoelectric point (pI) of proteins represents a parameter that can be predicted from genetic information as well as the * Corresponding author: (fax) +81-426-85-3742; (e-mail) shimurak@ pharm.teikyo-u.ac.jp. † Teikyo University. ‡ On leave from Agricultural University of Hebei, Baoding, P. R. China. § Laboratory of Molecular BioPhotonics. 10.1021/ac000387o CCC: $19.00 Published on Web 08/31/2000

© 2000 American Chemical Society

molecular mass. The determination of the pI of proteins is becoming increasingly important as the number of genes of known nucleotide sequences increases. For this determination, isoelectric focusing (IEF) in a pH gradient using a carrier ampholyte is currently the method of choice, especially in conjunction with a polyacrylamide-gel slab.1-3 Another possible option that can shorten the analysis time and reduce sample consumption is IEF in a capillary.4 In this procedure, protein samples can be detected in situ after the completion of focusing in a capillary5,6 or by passing the focused proteins through the detector.4,7,8 Ultraviolet absorption detection at 280 nm, which is based on absorption by protein tryptophan and tyrosine residues, is commonly used as the detection method. Although CIEF has already been used for the determination of the pI of proteins,9-12 critical evaluation on the accuracy of the determined values has been limited to the work by Kundu and Fenters,13 in which pI values of monoclonal antibodies were determined. In this paper, we report on a comprehensive evaluation of the accuracy of pI values determined by CIEF over a wide range of pH from the acidic to basic region that is usually used in IEF analyses. For this purpose, we chose nine proteins and a peptide as test samples. All of these compounds are in frequent use as pI markers in IEF analyses and, as a result, have been well characterized with respect to pI. Most commercial CE instruments are equipped with a stationary absorption detector and a mobilization process is required after the attainment of a steady-state conditions of IEF. In two-step CIEF, after focusing in a capillary with low electroosmosis, focused ampholytes are (1) Delince´e, H.; Radola, B. Anal. Biochem. 1978, 90, 609-623. (2) Låås, T.; Olsson, I.; So ¨derberg, L. Anal. Biochem. 1980, 101, 449-461. (3) Righetti, P. G. In Isoelectric Focusing: Theory, Methodology and Applications; Wood, T. S., Burdon, R. H., Eds.; Laboratory Techniques in Biochemistry and Molecular Biology 11; Elsevier Scientific: Amsterdam, 1983. (4) Hjerte´n, S.; Zhu, M.-D. J. Chromatogr. 1985, 346, 265-270. (5) Wang, T.; Hartwick, R. A. Anal. Chem. 1992, 64, 1745-1747. (6) Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 2934-2941. (7) Mazzeo, J. R.; Krull, I. S. Anal. Chem. 1991, 63, 2852-2857. (8) Thormann, W.; Caslavska, J.; Molteni, S.; Chmelı´k, J. J. Chromatogr. 1992, 589, 321-327. (9) Chen, S.-M.; Wiktorowicz, J. E. Anal. Biochem. 1992, 206, 84-90. (10) Schnabel, U.; Groiss, F.; Blaas, D.; Kenndler, E. Anal. Chem. 1996, 68, 4300-4303. (11) Lee, H. G. J. Chromatogr., A 1997, 790, 215-223. (12) Santora, L. C.; Krull, I. S.; Grant, K. Anal. Biochem. 1999, 275, 98-108. (13) Kundu, S.; Fenters, C. J. Capillary Electrophor. 1995, 2, 273-277.

Analytical Chemistry, Vol. 72, No. 19, October 1, 2000 4747

Table 1. Peptide pI Markers

b

no.

peptides

pIa

SDb

28 29 30 31 33 34 35 36 37 38 39 40 41 42 43

H-Trp-Tyr-Lys-Arg-OH H-Trp-Tyr-Lys-Lys-OH H-Trp-Tyr-Tyr-Lys-Lys-OH H-Trp-Tyr-Tyr-Tyr-Lys-Lys-OH H-Trp-Glu-Tyr-Tyr-Lys-Lys-OH H-Trp-Glu-His-His-His-Arg-OH H-Trp-Glu-His-Arg-OH H-Trp-Glu-His-His-OH H-Trp-Glu-Arg-OH H-Trp-Glu-His-OH H-Trp-Asp-Asp-His-His-OH H-Trp-Glu-Glu-His-OH H-Trp-Asp-Asp-Arg-OH H-Trp-Glu-Glu-OH H-Trp-Asp-Asp-Asp-OH

10.17 9.99 9.68 9.50 8.40 7.27 7.00 6.66 5.91 5.52 5.31 4.28 4.05 3.78 3.38

0.019 0.025 0.029 0.022 0.028 0.012 0.015 0.024 0.078 0.025 0.022 0.035 0.038 0.038 0.041

a Average of determined pI values of five independent experiments. Standard deviation of the determined pI values.

mobilized, along with a pH gradient, either by a pressure-driven hydrodynamic flow or by electrophoretic migration caused by the addition of a salt at one of the electrode solutions.4 On the other hand, focusing in a capillary via a mild electroosmosis permits a one-step CIEF in which a pH garadient is formed far from the detection point and ampholytes are focused before they pass the detection point by electroosmosis. In this paper, we adopted a two-step CIEF system, since it seems to be more flexible in terms of optimizing a focusing time. For mobilization, we chose a pressure-driven hydrodynamic flow, since an electrophoretic mobilization, in which ampholytes are detected, which are not in a real isoelectric state, might pose a bias, though it may be small, in the determination of pI. Another essential element for pI determination by CIEF is a set of standard substances that have known pI values, since analytical access to the small separation space in a capillary is quite restricted. Proteins, of known pI values, have been used for this purpose for some time. These pI marker proteins are not always satisfactory, because of their instability and tendency to become heterogeneous with respect to pI. The identification of the peak sometimes becomes ambiguous, and interpretation of results is subject to error. The detection protocols employed in CIEF permits the use of low-molecular-mass substances as pI markers. These have several advantages over macromolecular proteins, with respect to production, characterization, purity, and stability.14 We recently designed a set of peptides for use as pI markers and determined their pI values precisely.15 The set of peptide pI markers, each of which contains one tryptophan residue and two to five of the other amino acid residues contain an ionic side chain, formed very sharp peaks covering the pH range from 3.38 to 10.17 (Table 1). In this report, we used these markers in the determination of the pI of the test samples by CIEF using carrier ampholytes from different suppliers and in different pH ranges. The reevaluated pI values of these test samples showed good agreement with the values reported previously using alternate methods. The CIEF (14) Sˇ lais, K.; Fiedl, Z. J. Chromatogr. 1994, 661, 249-256. (15) Shimura, K.; Wang, Z.; Matsumoto, H.; Kasai, K. Electrophoresis 2000, 21, 603-610.

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proved to be another option for the accurate and precise determination of the pI of proteins and peptides. EXPERIMENTAL SECTION Materials. The following materials were obtained from commercial sources: Synthetic peptides (Peptide Institute, Inc., Minoh, Osaka, Japan); Pharmalyte, broad pI calibration kit (trypsinogen, pI 9.30; lentil lectins, pI 8.65, 8.45, 8.15; myoglobin, pI 7.35, 6.85; human carbonic anhydrase I, pI 6.55; bovine carbonic anhydrase II, pI 5.85; β-lactoglobulin A, pI 5.20; soybean trypsin inhibitor, pI 4.55; amyloglucosidase, pI 3.50) (Pharmacia Biotech AB, Uppsala, Sweden); Servalyt (Serva, Heidelberg, Germany); eCAP cIEF 3-10 kit (Beckman Instruments, Inc., Fullerton, CA); ribonuclease A (from bovine pancreas, pI 9.45), carbonic anhydrase II (from bovine erythrocyte, pI 5.90, formerly designated as B isozyme), β-lactoglobulin A (from bovine milk, pI 5.10), and CCK flanking peptide (included in eCAP cIEF kit, Beckman); trypsinogen (from bovine pancreas, pI 9.3), lentil lectin (from Lens culinaris, pI 8.8, 8.6, 8.2), myoglobin (from horse heart, pI 7.2, 6.8), carbonic anhydrase I (human erythrocyte, pI 6.6, formerly designated as B isozyme), carbonic anhydrase II (bovine erythrocyte, pI 5.9, formerly designated as B isozyme), β-lactoglobulin A (from bovine milk, pI 5.1), trypsin inhibitor (from soybean, pI 4.6), and amyloglucosidase (from Aspergillus niger, pI 3.6), hydroxypropylmethylcellulose (HPMC) (viscosity of 2% solution, 4000 cP at 25 °C) (Sigma Chemical Co., St. Louis, MO); fusedsilica capillaries (GL Sciences Inc., Tokyo). CIEF. A cIEF kit, obtained from Beckman, was operated according to the manufacturer’s recommendations using an automated capillary electrophoresis instrument (Beckman P/ACE 2210 with a UV detector). An eCAP neutral capillary (50 µm i.d. × 27 cm) was rinsed with water for 1 min by the high-pressure mode (20 psi or 1.4 atm) and then filled with the ampholyte gel solution containing the test samples for 1 min using the highpressure mode. A mixture of the peptide marker solution (0.25 mM each peptide) was injected from the anodic side for 10-30 s by the injection mode (0.5 psi or 0.035 atm). Focusing was carried out at 500 V/cm for 2 min at 25 °C with 91 mM phosphoric acid in the cIEF gel as the anolyte and 20 mM NaOH as the catholyte. To mobilize the focused peptide zones through the detector, which was located at 7 cm from the cathodic end, pressure was applied at the anodic end by the low-pressure rinse mode, while maintaining a field strength of 500 V/cm. The proteins and peptides were detected by measurement of the absorption at 280 nm. Other CIEF experiments were carried out with the same instrument basically following the reported procedure.16 Fusedsilica capillaries (50 µm i.d., 375 µm o.d., 27 cm long) were internally coated with linear polyacrylamide17 and rinsed with water for 15 min by the high-pressure rinse mode, before use for the first time. Pharmalyte or Servalyt was used at a 40-fold dilution (∼1% w/v) from original solutions (∼40% w/v) with the addition of HPMC at a concentration of 0.2-0.4% (w/v) and N,N,N′,N′tetramethylethylenediamine (TEMED) at a concentration of 0.10.3% (v/v) according to the specific electrophoresis conditions. This solution is referred to as the 1× CA solution. The catholyte was 20 mM NaOH and the anolyte was 20 mM phosphoric acid (16) Huang, T.-L.; Shieh, P. C. H.; Cooke, N. Chromatographia 1994, 39, 543548. (17) Hjerte´n, S. J. Chromatogr. 1985, 347, 191-198.

except for the pH range 5-10.5, where 20 mM L-glutamic acid was used as anolyte. HPMC was added to the anolyte at a concentration of 0.1-0.4%, to control the mobilization speed of the anodic side of the pH gradient. The polarity was set as the anode to be the inlet with a distance of 20 cm between the anodic end and the detection window and the temperature was set at 25 °C. The capillary was washed with water in the high-pressure rinse mode for 1 min. Test samples were dissolved in 1× CA solution at a concentration of 0.02-0.1 µg/µL for each protein in most cases, and the solution was injected into the capillary from the anodic end at the high-pressure rinse mode for 1 min to fill the capillary with the solution. Peptide pI markers, dissolved in water, were injected from the anodic end for 10-50 s by the injection mode. The pH of the peptide pI marker solution was ∼3.5, and this level of acidity was important for the stability of the markers.15 Isoelectric focusing was initiated by applying an electric field of 500 V/cm. At 2-20 min after the initiation, pressure was applied at the anodic end by the lowpressure rinse mode (0.5 psi) to start the mobilization of the pH gradient toward the cathode, while maintaining a constant voltage. The proteins and peptide pI markers were detected by absorption at 280 nm at 7 cm from the cathode. The isoelectric focusing was finished before the current went up to 10 µA. The isoelectric points of the test samples were determined from the detection time of their peaks by assuming a linear relationship between the pH and detection time within a pH range flanked by two peptide pI markers. Determination of the pI of Proteins by Slab Gel IEF. The isoelectric points of bovine trypsinogen and lentil lectin were determined by means of an IEF experiment using polyacrylamide gel slabs basically following the procedure for the determination of the pI of the peptide pI markers.15 A polyacrylamide gel slab (5%T, 3%C, 55 × 115 × 1 mm) containing 10% glycerol and Pharmalyte 8-10.5 at a 16-fold dilution of a purchased solution was prepared on a glass plate without TEMED. Ammonium persulfate was used at a final concentration of 0.05% (w/v), to initiate the polymerization. On the backside of the plates, lines were drawn at 1-cm intervals parallel to the shorter edges of the gel with a water-resistant pen as positional markers to correlate the results of the pH measurement of the gel after completion of focusing and the electropherogram obtained by Coomassie Blue staining. The proteins were applied though a sample application mask and focusing was carried out at 250 V for 15 min, 500 V for 15 min, and 1000 V for 3 h with a 10-cm distance between the electrodes at 25 °C with a coolant temperature of 20 °C. The pH of the gel was measured at 25 °C on each line drawn on the back of the plate with a portable pH meter (product No. HW17MX; Toa Electronics, Tokyo) with a Metox pH sensor (product No. MTX-6101F; Toa Electronics). The gel was stained according to the procedure described in the instruction of the pI calibration kit from Pharmacia; the pIs of the focused points were determined by assuming a linear relationship between pH and position within two lines, above which the pH of the gel was measured. Evaluation of the Validity of the Determined pI Values by CIEF. The difference between the determined and the reference pI value of a test sample was used as a criterion for the validity of the pI determination by CIEF. Most of the pI values reported by Pharmacia and Sigma are not in agreement. In such cases, the

average of the two reported values was used (Table 2). For trypsinogen, lentil lectin, and CCK flanking peptide, the values determined by us, using slab gel IEF were used as reference values. For trypsinogen, our value was 9.31, which was close to the suppliers’ value of 9.3. Our values for three pI variants of lentil lectin, 8.66, 8.42, and 8.11, were close to those reported by Pharmacia, 8.65, 8.45, and 8.15, respectively. The reported value for CCK flanking peptide by Beckman is apparently erroneous, and our value, 3.67, was used. For ribonuclease A, Beckman reports 9.45, a value that is presumably based on values reported by Anderson and Alberty in 1948,18 but we adopted a more recent value reported by Tanford and Hauenstein in 1956 as the isoionic point, 9.60.19 Theoretically, the pI value should be higher than the isoionic point at this pH (see Results and Discussion). RESULTS AND DISCUSSION Test Samples for pI Determination by CIEF. As test samples, nine proteins and a peptide, which have previously been used as pI markers, were chosen. These proteins are generally assumed to be well characterized with respect of their isoelectric points (Table 2). The proteins were collected from three suppliers. Pharmacia Biotech produces a mixture of proteins for the purpose of calibration of a pH gradient formed in IEF. Sigma Chemical Co. supplies each protein separately as IEF markers, and they were used individually or in the form of a mixture in the experiments in this report. The pI marker proteins and CCK flanking peptide from Beckman were included in the cIEF kit of Beckman as separate samples. The reported values by the suppliers for these proteins are listed in the Materials section. There is a small discrepancy between the values reported by the different suppliers for the same protein with the largest difference of 0.15 pH unit for the two basic species of lentil lectin and the basic species of myoglobin. In such cases, an average value was used as the reference value. On the other hand, amyloglucosidase appeared as two relatively broad peaks in CIEF, even though two suppliers report a single but different pI value for this protein as 3.5 and 3.6, respectively. We adopted the average of the two as the reference value, 3.55, and the determined pI value for each peak in CIEF was compared with this number. The test samples are listed in Table 2, along with their reference values. In the early experiments, we found a significant discrepancy between the determined values for the isoforms of lentil lectin by CIEF and the values reported by suppliers. As reported previously,15 we found an error in the reported value, 2.75, for the CCK flanking peptide by the supplier and determined its pI to be, in fact, 3.67. We suspected some errors in the values reported for the isoforms of lentil lectin or some changes in pI values of a particular preparation of a protein from those reported by the suppliers even before starting this study. We, thus, undertook the redetermination of the pI values of trypsinogen and lentil lectin by slab gel IEF. Our determination, however, were in agreement with the values reported by Pharmacia within a range of experimental errors and showed a small discrepancy with those reported by Sigma for lentil lectin with the difference of -0.14 pH unit at the largest. For trypsinogen and lentil lectin, the values determined by us were used as the reference values. (18) Anderson, E. A.; Alberty, R. A. J. Phys. Colloid Chem. 1948, 52, 13451364. (19) Tanford, C.; Hauenstein, J. D. J. Am. Chem. Soc. 1956, 78, 5287-5291.

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Table 2. Summary of the Determination of pIs of Proteins Using CIEF with Peptide pI Markers under Different Conditionsa pI values determined by CIEFb

proteins

origins

ref pI values

ribonuclease A

bovine pancreas

9.60c

trypsinogen

bovine pancreas

9.31e

lentil lectin

lentil

8.66e

lentil lectin

lentil

8.42e

lentil lectin

lentil

8.11e

myoglobin

horse heart

7.28

myoglobin

horse heart

6.83

carbonic anhydrase I

human erythrocyte

6.58

carbonic anhydrase II bovine erythrocyte

5.88

β-lactoglobulin A

bovine milk

5.15

trypsin inhibitor

soybean

4.58 3.67f

CCK flanking peptide amyloglucosidase

Aspergillus niger

3.55

amyloglucosidase

Aspergillus niger

3.55

av of absolute values of the differences

Beckman cIEF kit exp 1 (5)

Beckman cIEF kit exp 2 (4)

Pharmalyte Pharmalyte 5-10.5 (3) 2.5-8 (3)

Servalyte 3-7 (3)

9.43 0.03/-0.17

7.27 0.00/-0.01 6.86 0.00/+0.03 6.45 0.01/-0.13 5.84 0.01/-0.04 5.24 0.02/+0.09 4.45 0.01/-0.13 3.53 0.03/-0.14 3.48 0.02/-0.07 3.44 0.01/-0.11 0.083

9.36 9.25 0.01/+0.05 0.01/-0.06 8.63 0.01/-0.03 8.4 0.00/-0.02 8.03 0.01/-0.08 7.23 7.27 0.03/-0.05 0.00/-0.01 6.83 peak split 0.00/0.00 6.4 6.63 0.00/-0.18 0.01/+0.05 5.83 5.93 0.01/-0.05 0.02/+0.05 5.22 0.03/+0.07 4.57 0.01/-0.01 3.45 0.01/-0.10 3.42 0.01/-0.13 0.071

0.043

Pharmalyte 3-10 (3)

Servalyte 3-10 (3)

9.68 0.00/+0.08 9.31 0.00/0.00 8.68 0.01/+0.02 8.4 0.00/-0.02 7.94 0.00/-0.17 7.27 0.00/-0.01

IFd

0.073

0.046

IFd

5.27 0.01/+0.12 4.74 0.01/+0.16 3.65 0.01/-0.02 3.6 0.01/+0.05 3.53 0.01/-0.02

8.69 0.01/+0.03 8.4 0.00/-0.02 7.98 0.00/-0.13 7.27 0.00/-0.01 6.83 0.02/0.00 6.59 6.66 0.00/+0.01 0.00/+0.08 5.91 5.91 0.00/+0.03 0.00/+0.03 5.21 5.22 5.18 0.02/+0.06 0.01/+0.07 0.02/+0.03 4.64 4.96 4.44 0.02/+0.06 0.04/+0.38 0.02/-0.14 3.71 3.68 3.67 0.01/+0.04 0.01/+0.01 0.01/0.00 3.66 not detected 3.6 0.01/+0.11 0.01/+0.05 3.56 not detected 3.52 0.01/+0.01 0.01/-0.03

0.074

0.056

aThe first line in each cell is the average of three to five experiments. The second line is the standard deviation of the determination and, after slash, the difference from a reference value b The determined values are the average of the multiple experiments with the number of experiments in the parentheses. c The value is an isoionic point determined by Tanford and Hauenstein.19 d Imperfect focusing. e The values were determined by quadriplicate IEF experiments in polyacrylamide-gel slabs using Pharmalyte 8-10.5 at 25 °C. Standard deviation was 0.05 for all values. f The value was determined by triplicate IEF experiments in polyacrylamide-gel slabs using Pharmalyte 2.5-5 at 25 °C. Standard deviation was 0.02.

The fact that two lentil lectin preparations from Sigma showed different patterns on slab gel IEF caused some confusion. One product, L1277, listed as IEF markers showed three bands with pIs of 8.66, 8.42, and 8.11 as shown in Table 2. Another product, L9267, listed in the section on lectins appeared as two bands with pI values of 9.06 and 8.70 as the average of quadruple experiments with standard deviations of 0.024 and 0.010, respectively. The lower pI variant in L9267 appeared to have the same pI as the highest pI variant of L1277. This situation represents one of the problems in the use of proteins as pI markers; i.e., preparations of the same protein can have different pI values and the origin of the difference is not always easy to assign. L1277 was used as lentil lectin in the experiments described below. Use of Commercial Kit Designed for the Instrument. A kit that is specially designed for CIEF with the instrument is available from Beckman. We initially tested the kit for the determination of pI of the test samples. Samples were included in the ampholyte gel solution and the capillary was filled with the solution. A mixture of the 15 peptide pI markers was injected at the anodic end, and the focusing was started. The separate injection of the pI markers allowed more flexibility in changing the size of the peaks of pI markers, which were superimposed to those of the test samples and facilitated the identification of the origin of peaks, from either a sample or the markers. Two minutes 4750 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

later, a pressure of 0.5 psi was applied at the anodic end to mobilize the focused proteins, while the electric field was maintained. The electropherogram for the pI markers alone is shown in Figure 1A and that obtained by cofocusing of the test samples with the markers is shown in Figure 1B. The most basic two peptide pI markers, No. 28 (pI 10.17) and 29 (pI 9.99) were not resolved under these conditions. The sample contained ribonuclease A, myoglobin, carbonic anhydrase I, carbonic anhydrase II, β-lactoglobulin A, trypsin inhibitor, CCK flanking peptide, and amyloglucosidase. That the detection time of the peaks was not fully reproducible is probably due to subtle changes in the electroosmotic flow of the capillary. The peaks were readily identified by comparison of relative positions of the peaks between the two electropherograms and those obtained by some additional experiments with a single sample. The pH profile of the pH gradient in this system was obtained by plotting pI of markers against the detection time of each peak for the experiment in Figure 1B (Figure 2). As can be seen in the close appearance of the peaks of basic peptide markers, the basic end of the gradient was very steep and the acidic end was shallow. As a result, the pH profile was curved and the representation of the pH gradient by a single straight line was found to be invalid and irrelevant, although it is recommended by the supplier of the kit. It was found that the simple replacement of the ampholyte

Figure 1. CIEF of the set of peptide pI markers and test samples using the Beckman cIEF 3-10 kit. Test samples were dissolved in the ampholyte gel solution at the following concentrations: ribonuclease A (RN) 350 µg/mL, myoglobin (MGb and MGa (b and a for basic and acidic variants, respectively) 20 µg/mL, carbonic anhydrase I (CAI) 40 µg/mL, carbonic anhydrase II (CAII) 30 µg/mL, β-lactoglobulin A (LG) 60 µg/mL, trypsin inhibitor (TI) 40 µg/mL, CCK flanking peptide (CCK) 20 µg/mL, and amyloglucosidase (AG) 40 µg/mL. An eCAP neutral capillary (50 µm i.d. × 27 cm) was installed in a Beckman P/ACE 2210 with a UV detector with the polarity of the inlet side to the anode. The capillary was filled with the ampholyte gel solution which contained the samples and the mixture of the 15-peptide marker solution was injected from the anodic side by the injection mode (0.5 psi). Focusing was carried out at 500 V/cm for 2 min at 25 °C with 91 mM phosphoric acid in the cIEF gel as anolyte and 20 mM NaOH as catholyte. To mobilize the focused protein and peptide zones, pressure (0.5 psi) was applied at the anodic end, while a field strength of 500 V/cm was maintained. The proteins and peptides were detected by absorption at 280 nm. The electric current is indicated by dotted lines. (A) Mixture of the pI markers alone (0.25 mM each, 30 s injection); (B) test samples and pI markers (30 s injection). A part of the electropherogram of (B) from 8.5 to 10.5 min was expanded in the direction of detection time and included as an inset of (B). The numbers put above the peaks are the peptide number listed in Table 1.

Figure 2. pH profile for the Beckman cIEF 3-10 kit. The pI of each peptide pI markers on the experiment of Figure 1B were plotted against detection time of each marker. Each point on the plot is connected by straight lines.

gel solution between the inlet and the detection window by the anolyte using the low-pressure rinse mode did not require more than 15 min, and therefore, the delay of the mobilization of the

acidic end of the pH gradient seems somehow to be related to an electrophoretic phenomenon. One possible cause for this delay is the anodic drift of the pH gradient, i.e., the electrophoretic migration of negatively charged carrier ampholyte in the acidic end of the pH gradient. Acidic carrier ampholytes are slightly negatively charged in focusing conditions in order to maintain electric neutrality with the hydrogen ion whose concentration gradually increases at the acidic end.3 The nonlinear relationship in pI vs detection time plots was also pointed out in the application of one-step CIEF, and the use of a fitted nonlinear standard curve for a relatively small number of pI markers was found to improve accuracy in the pI determination of monoclonal antibodies.13 Since the peptide pI markers used in this report scatter well over the pH range of IEF analyses with relatively narrow gaps, we thought that simple connection of consecutive marker points with straight lines would be more straightforward to represent the pH gradient in a capillary than the use of a fitted curve. The pI values of the samples were, thus, calculated by assuming a linear relation of pH against detection Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

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time between the two flanking marker peptides. The averages of the results of five experiments are listed in Table 2 in the column of Beckman cIEF kit exp 1 with standard deviations and the differences from reference values. The standard deviations of the consecutive determinations were 0.03, at most. For three proteins and one peptide, the absolute values of the differences were larger than 0.1 but, on average, the absolute value of the differences was 0.083. Ribonuclease was observed slightly behind the No. 31 marker (pI 9.50) (inset of Figure 1B) and its pI was determined to be 9.43. The isoelectric point of ribonuclease A has been reported to be 9.45 by Anderson and Alberty,18 and Beckman prints the same value on the label of the sample protein. On the other hand, an isoionic point of 9.604 (ionic strength 0.001) was reported by Tanford and Hauenstein more recently.19 We adopted this value as the reference and hence the determined pI was 0.17 pH unit lower than the reference value. At this pH, hydroxide ions are in excess in the solution and the protein at these isoionic conditions has a small positive charge; i.e., the isoelectric point should be slightly higher than the isoionic point, although the difference is small. A different series of experiment was carried out under the same conditions as described above by using the broad pI calibration kit from Pharmacia as a test sample. This protein pI marker kit contains trypsinogen and lentil lectin instead of ribonuclease A and CCK flanking peptide, which were contained in the mixture used in the previous series of experiments. Trypsinogen was not detected when this kit was analyzed and the peaks for lentil lectin were not clearly identifiable. For carbonic anhydrase I and amyloglucosidase, smaller values of more than -0.1 pH unit were obtained in comparison with the reference values (Table 2, column for the Beckman cIEF kit exp 2). For the other five proteins, the absolute values of the difference from the reference became smaller than 0.1 in this series of experiments. The pI value of trypsinogen was determined separately and good agreement was observed. A relatively large difference of 0.12 pH unit was observed in the two determinations for trypsin inhibitor. The pI value of this protein, as determined by CIEF, fluctuated throughout the experiments reported below. The fluctuation may be related to the nature of this protein. For other samples, the difference between the two series of experiments was less than 0.05 pH unit, showing satisfactory reproducibility. Use of a Mixture of Pharmalyte pH 5-8 and 8-10.5. To improve the precision of the pI determination by CIEF, a narrower pH range carrier ampholyte was tested (Figure 3). As a carrier ampholyte, Pharmalyte 5-8 and 8-10.5 were both mixed at an 80-fold dilution, resulting in a total 40-fold dilution. TEMED was included in the 1× CA solution at a concentration of 0.1% as a spacer between the catholyte and the basic end of pH gradient, and HPMC was added at a concentration of 0.4% to control the mobilization speed. The capillary coated with polyacrylamide was filled with a 1× CA solution, which contained the test samples. The mixture of the peptide pI markers was injected at the anodic end for 30 s, and focusing was initiated at 500 V/cm with 20 mM L-glutamic acid containing 0.4% HPMC as the anolyte and 20 mM NaOH as the catholyte. After 2 min, mobilization was started by pressurizing at the anode, while constant voltage was maintined. It was found that, when the coated capillary was used for the first 4752 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

Figure 3. CIEF of the peptide pI markers and protein samples using a mixture of Pharmalyte 5-8 and 8-10.5 as carrier ampholyte. The composition of the 1× CA solution was Pharmalyte 5-8 at 80-fold dilution, Pharmalyte 8-10.5 at 80-fold dilution, 0.1% TEMED, and 0.4% HPMC. Protein samples were dissolved in the 1× CA solution at the following concentrations: trypsinogen (TG) 100 µg/mL; lentil lectin (LLb, LLi, and LLa (b, i, and a for basic, intermediate, and acidic variants, respectively) 200 µg/mL. A fused-silica capillary (50 µm i.d. × 27 cm) coated with linear polyacrylamide was used. Other conditions were the same as those for the experiments shown in Figure 1, except that the composition of the anolyte was 20 mM L-glutamic acid containing 0.4% HPMC. (A) pI markers alone (30 s injection); (B), protein samples and the pI markers (30 s injection); (C) protein samples alone. The numbers above the peaks are the peptide numbers listed in Table 1.

time, thorough washing with water was important, otherwise an abnormal separation was observed. More than a 15-min wash with water by the high-pressure rinse mode is recommended.

Figure 4. pH profile for CIEF with Pharmalyte 5-8 and 8-10.5. The pI of each peptide pI marker in the experiment of Figure 3B was plotted against the detection time of each marker. Each point on the plot is connected by straight lines.

Although the pH range of the carrier ampholyte was 5-10.5, focusing was observed for No. 40 pI marker (pI 4.28) and those having even lower pI values (data not shown). This should be due to the use of an ampholyte, glutamic acid, as the anolyte. Trypsinogen and lentil lectin was subjected to the CIEF separation (Figure 3C). Trypsinogen appeared at ∼9 min with two associated peaks on both sides. Lentil lectin appeared at 10-13 min in the form of a triplet. Two sharp peaks of unknown origin were observed at 17-21 min. The determination of pI was carried out by cofocusing of the test samples and pI markers (Figure 3B). Although there were some variations in detection time, the peaks were readily identified by comparing the electropherograms with respect to the relative position and peak height. The pH profile of the experiment shown in Figure 3B is shown in Figure 4. The two most basic pI markers were not resolved, even by extending the pH range of the carrier ampholyte to pH 10.5, and a sigmoidal curvature was observed at pH 6-7. The determined pI values for trypsinogen and lentil lectin are listed in Table 2 in the column of Pharmalyte 5-10.5 showed very small differences from the reference values. Myoglobin, carbonic anhydrase I, and carbonic anhydrase II were also subjected to pI determination by using the same carrier ampholyte as above except for the absence of TEMED in the 1× CA solution (Figure 5). The basic variant of myoglobin overlapped with the No. 34 pI marker (pI 7.27), carbonic anhydrase I appeared slightly behind the No. 36 pI marker (pI 6.66), and carbonic anhydrase II emerged slightly before the No. 37 pI marker (pI 5.91) (Figure 5B). Two small peaks emerged at the position where the acidic pI variant of myoglobin would be expected to appear (18-19 min). Further identification of the peaks was not carried out, and the attempt to determine the pI of the acidic variant was abandoned. Good agreement was found between the determined pI values by the CIEF experiment and the reference values. Even though the two most basic pI markers were not separated under these conditions, the first marker peak was suspiciously small. The detection sensitivity might be decreased at the cathodic end of the pH gradient, for unknown reason. Use of a Mixture of Pharmalyte pH 2.5-5 and 5-8. For the determination of pI values of acidic proteins, a mixture of Pharmalyte 2.5-5 and 5-8 was used. Two carrier ampholytes were mixed both at 80-fold dilution, resulting in a total 40-fold

Figure 5. CIEF of the peptide pI markers and protein samples using a mixture of Pharmalyte 5-8 and 8-10.5 as carrier ampholyte. Protein samples were dissolved in the 1× CA solution at the following concentrations: myoglobin (MGa and MGb) 20 µg/mL, carbonic anhydrase I (CAI) 40 µg/mL, and carbonic anhydrase II (CAII) 30 µg/mL. Other conditions were the same as those for the experiments shown in Figure 3 except for the absence of TEMED in the 1× CA solution. (A) pI markers alone (30 s injection); (B) protein samples and the pI markers (30 s injection); (C) protein samples alone. The numbers above the peaks are the peptide numbers listed in Table 1.

dilution. The 1× CA solution contained 0.4% HPMC but no TEMED. The anolyte was 20 mM phosphoric acid containing 0.2% HPMC, and the catholyte was 20 mM NaOH. A polyacrylamide-coated capillary was used, as described in the previous section. The pI marker was injected at the anode and focused at a voltage of 500 V/cm. In preliminary experiments, the pressure mobilization was initiated after 2 min of focusing and an anolyte that contained HPMC at 0.4% was used. Under these conditions, the most acidic marker (No. 43, pI 3.38) was not detected before 46 min. Neither an increase in the concentration of phosphoric acid to 200 mM nor the use of sulfuric acid at 200 mM alleviated this problem. A decrease in the concentration of HPMC in the anolyte to 0.2% was effective in canceling the elongation of the detection time of the acidic end of the pH gradient and No. 43 marker became detectable at 35 min. β-Lactoglobulin A, trypsin inhibitor, CCK-flanking peptide, and amyloglucosidase were subjected to the analysis using 20 mM phosphoric acid containing 0.2% HPMC as anolyte (Figure 6). When mobilization was initiated at 2 min after the start of focusing, anomalous pI values of 5.9 and 4.9 were obtained for β-lactoAnalytical Chemistry, Vol. 72, No. 19, October 1, 2000

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Figure 6. CIEF of the set of peptide pI markers and protein samples using a mixture of Pharmalyte 2.5-5 and 5-8 as carrier ampholyte. The composition of the 1× CA solution was Pharmalyte 2.5-5 at an 80-fold dilution, Pharmalyte 5-8 at an 80-fold dilution, and 0.4% HPMC. Test samples were dissolved in 1× CA solution at the following concentrations: β-lactoglobulin (LG) 60 µg/mL, trypsin inhibitor (TI) 30 µg/mL, CCK franking peptide (CCK) 20 µg/mL, and amyloglucosidase (AG) 40 µg/mL. A fused-silica capillary (50 µm i.d. × 27 cm) coated with a linear polyacrylamide was used. Other conditions are the same as those for the experiments of Figure 1 except for the composition of the anolyte, 20 mM phosphoric acid containing 0.2% HPMC, and the focusing time of 20 min. The pI marker solution (0.25 mM each) was injected from anodic end for 15 s. The numbers above the peaks are the peptide numbers listed in Table 1. Inset is the pH profile of the experiment.

globulin A and trypsin inhibitor, respectively, and the peak for the trypsin inhibitor was accompanied with broad skirt especially spreading toward the basic side. An increase in the focusing time shifted the peaks of the two proteins toward the acidic side and a steady state was nearly reached at 20-min focusing prior to the start of mobilization (Figure 6). The determined pI values were listed in Table 2 in the column Pharmalyte 2.5-8. The values for β-lactoglobulin A and trypsin inhibitor were a little higher than the reference values with a difference of +0.12 and +0.16, respectively, but the agreement for CCK flanking peptide and amyloglucosidase was very good. Care should be taken in attaining the steady state under the experimental conditions used. This can be verified by the consistency of the pI value determined by an additional experiment with a different focusing time. Use of Servalyt pH 3-7. The same sample as that used in the previous section was analyzed by CIEF using Servalyt 3-7 as a carrier ampholyte (Figure 7). The 1× CA solution contained 1% Servalyt 3-7 (40-fold dilution) and 0.4% HPMC but no TEMED. The anolyte was 20 mM phosphoric acid with 0.1% HPMC, and the catholyte was 20 mM NaOH. Other conditions were the same as those for the experiments in the previous section. Focusing was carried out at 500 V/cm for 20 min, and pressure mobilization was initiated. With this carrier ampholyte, the decrease of the concentration of HPMC to 0.2% in the anolyte was still not sufficient and the curvature of the pH profile was still evident (open diamonds of the inset of Figure 7). A further decrease in HPMC concentration to 0.1% led to a more linear pH profile (closed circles of the inset of Figure 7). β-Lactoglobulin and trypsin inhibitor appeared as broad bands, probably due to a degree of microheterogeneity in these protein samples with respect to isoelectric point. The peaks for the CCK flanking peptide and amyloglucosidase were sharper in comparison to those obtained by CIEF using Beckman 3-10 kit or Pharmalyte 2.5-8. The results of the pI 4754 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

Figure 7. CIEF of the set of peptide pI markers and protein samples using Servalyt 3-7 as carrier ampholyte. The composition of the 1× CA solution was 1% Servalyt 3-7 (40-fold dilution) and 0.4% HPMC. Test samples were dissolved in the 1× CA solution at the following concentrations: β-lactoglobulin (LG) 40 µg/mL, trypsin inhibitor (TI) 30 µg/mL, CCK franking peptide (CCK) 20 µg/mL, and amyloglucosidase (AG) 40 µg/mL. A fused-silica capillary (50 µm i.d. × 27 cm) coated with linear polyacrylamide was used. Other conditions are the same as those for the experiments of Figure 1 except for the composition of the anolyte, 20 mM phosphoric acid containing 0.1% HPMC, and the focusing time of 20 min. The pI marker solution (0.25 mM each) was injected from the anodic end for 40 s. The numbers above the peaks are the peptide numbers listed in Table 1. Inset is the pH profile of the experiments obtained with the anolytes containing HPMC at concentrations of 0.1 (b) or 0.2% (]).

determination are summarized in the column Servalyt 3-7 in Table 2. The differences from the reference values were smaller than 0.1 for all samples, when the differences for amyloglucosidase were averaged. Use of Wide pH Range Pharmalyte and Servalyt. When two commercially available wide-range carrier ampholytes, Pharmalyte 3-10 and Servalyt 3-10, were tested for CIEF with HPMC in the 1× CA solution and the anolyte both at 0.4%, the nonlinear delay in the detection of acidic peptide pI markers was again observed as was experienced with pH gradients extending to pH 3. We tested several concentrations of HPMC in the anolyte, 20 mM phosphoric acid, and found that 0.1% (w/v) was the optimum, in combination with 1× CA solution containing 0.4% HPMC. The determination of the pI of test samples was carried out with the two carrier ampholytes. Pharmalyte pH 3-10 was used at a 40-fold dilution of the original solution (∼40% (w/v)) as the 1× CA solution containing 0.3% TEMED and 0.4% HPMC. The catholyte was 20 mM NaOH, and the anolyte was 20 mM phosphoric acid containing 0.1% HPMC. A polyacrylamide-coated capillary was used, and CIEF was carried out as described in the Experimental Section with a focusing time of 10 min. The electropherogram for the set of peptide pI markers is shown in Figure 8A. The most basic two pI markers, No. 28 (pI 10.17) and No. 29 (pI 9.99) were separated for the first time. The pH profile obtained by plotting pI against detection time curved between pH 4 and 6 (Figure 9). The region for the No. 37-39 markers was condensed corresponding to the steep pH profile from pH 5 to 6. By way of example, the electropherograms for trypsinogen and lentil lectin are shown in Figure 8B-E. The values determined by CIEF are listed in the column corresponding to Pharmalyte 3-10 in Table 2. A relatively large

Figure 8. CIEF of the set of peptide pI markers and sample proteins using Pharmalyte 3-10 as carrier ampholyte. The composition of the 1× CA solution was 1% Pharmalyte 3-10 (40-fold dilution), 0.3% TEMED, and 0.4% HPMC. Protein samples were dissolved in the 1× CA solution at the following concentrations: trypsinogen (TG) 50 µg/mL and lentil lectin (LLb, LLi, and LLa) 160 µg/mL. A fused-silica capillary (50 µm i.d. × 27 cm) coated with linear polyacrylamide was used. Other conditions were the same as those for the experiments described in Figure 1 except for the composition of the anolyte, 20 mM phosphoric acid containing 0.1% HPMC, and the focusing time of 10 min. (A) pI markers (0.25 mM each, 50 s injection); (B) trypsinogen; (C) trypsinogen and the pI markers (30 s injection); (D) lentil lectin; (E) lentil lectin and the pI markers (30 s injection). The numbers above the peaks are the peptide numbers listed in Table 1.

Figure 9. pH profile for CIEF with Pharmalyte 3-10. The pI of each peptide pI marker in the experiment of Figure 8A was plotted against detection time. Each point on the plot is connected by straight lines.

discrepancy was observed for the acidic pI variant of lentil lectin and trypsin inhibitor; i.e., a difference from the reference value of -0.17 and +0.38, respectively, was observed. The difference

for trypsin inhibitor was exceptionally large. This may be due to the short focusing time, as was evident in the experiments using Pharmalyte 2.5-8, or the assumption of linear relationship may not be valid in this portion of the pH gradient (Figure 9). Amyloglucosidase could not be detected under the experimental conditions even though the most acidic peptide pI marker (No. 43, pI 3.38), the pI of which is lower than that of amyloglucosidase, was detected without any problem. Servalyt 3-10 was tested at a 40-fold dilution of the original solution (40% (w/v)) as the 1× CA solution containing 0.3% TEMED and 0.4% HPMC. The other conditions of CIEF were the same as those for Pharmalyte 3-10 described above. The focusing pattern of the 15 peptide pI markers is shown in Figure 10A. The most basic two peptides were not resolved. The pH profile also showed a curvature between pH 4 and 5 (Figure 11) but it was less prominent than that observed for Pharmalyte 3-10. The determined pI values for the test samples are listed in the column of Servalyt 3-10 in Table 2. The absolute values of the differences were largely less than 0.1 except for the acidic pI variant of lentil Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

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Figure 10. CIEF of the set of peptide pI markers and protein samples using Servalyt 3-10 as carrier ampholyte. The composition of the 1× CA solution was 1% Servalyt 3-10 (40-fold dilution), 0.3% TEMED, and 0.4% HPMC. Test samples were dissolved in 1× CA solution at the following concentrations: myoglobin (MGa and MGb) 20 µg/mL, carbonic anhydrase I (CAI) 40 µg/mL, carbonic anhydrase II (CAII) 30 µg/mL, β-lactoglobulin (LG) 60 µg/mL, trypsin inhibitor (TI) 30 µg/mL, CCK flanking peptide (CCK) 20 µg/mL, and amyloglucosidase (AG) 40 µg/mL. A fused-silica capillary (50 µm i.d. × 27 cm) coated with linear polyacrylamide was used. Other conditions were the same as those for the experiments of Figure 1 except for the composition of the anolyte, 20 mM phosphoric acid containing 0.1% HPMC, and the fact that a focusing time of 10 min was used. (A) pI markers (0.25 mM each, 30 s injection); (B) test samples and the pI markers (30 s injection). The numbers above the peaks are the peptide numbers listed in Table 1.

Figure 11. pH profile for CIEF with Servalyt 3-10. The pI of each peptide pI marker in the experiment of Figure 10A was plotted against detection time. Each point on the plot is connected by straight lines.

lectin and trypsin inhibitor, which had values of-0.13 and -0.14, respectively. Ribonuclease A and trypsinogen appeared as two peaks with this carrier ampholyte under the experimental conditions used (data not shown). A slow focusing of the proteins under these conditions may be the cause of the two peaks, but a simple extension of the focusing time compromised the separation at the basic end and did not solve the problem. An example of an electropherogram for the determination of pI of some proteins is shown in Figure 10B. The shoulder peak of the trypsin inhibitor may be due to imperfect focusing. Because of the overlapping of the CCK flanking peptide and the basic variant of amyloglucosidase, the pI values of the two samples were determined separately. Under the experimental conditions used, the most acidic protein, amyloglucosidase, was not detected with Pharmalyte. On 4756

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the contrary, two basic protein, ribonuclease A and trypsinogen, were not well focused with Servalyt 3-10. To solve the problems, a mixture of equal amounts of the 1× CA solutions based on Pharmalyte 3-10 and Servalyt 3-10 was tested. However, the problems were reproduced at both extremes of the pH gradient. The use of a mixture of three narrow-range Pharmalyte solutions, 2.5-5, 5-8, and 8-10.5, gave the same result as Pharmalyte 3-10 (data not shown). Up to this point, the peptide pI markers were loaded into the capillary by injection from the anode. Injection from the cathode or inclusion of the markers in the 1× CA solution was also tested. Essentially the same results were obtained with the loading from the anode when Pharmalyte 3-10 was used (data not shown). A difference was observed within 4 min after the start of focusing, where small and closely separated peaks were detected, which corresponded to migrating markers from the cathodic side to focusing points. On the other hand, with Servalyt 3-10, when the markers were included in the 1× CA solution, the peak heights of the five most basic peptides were reduced and two unidentified peaks appeared at the basic end. When the markers were loaded from the cathode, the five peaks were no longer detected and the two unidentified peaks again appeared. Some interactions between basic peptide pI markers and Servalyt 3-10 might have occurred, when the entire column or the cathodic loading of the markers was used. The same type of interactions might be involved in the problems with ribonuclease A and trypsinogen. Since the effectiveness of these carrier ampholytes has been thoroughly tested for their use in slab gel IEF, the observed problems might be related to specific experimental conditions used in our CIEF experiments.

CONCLUSION The pI determination of nine proteins and one peptide by CIEF was carried out with a set of recently developed peptide pI markers under a variety of experimental conditions with respect to the source of carrier ampholyte and the range of the pH gradient. The sharp peaks of the highly pure peptide pI markers were unambiguously identified when they were cofocused with test samples. The averages of the absolute values of the differences were calculated for each experiment and are listed in the bottom line of Table 2 ranging from 0.043 to 0.083. This indicates that the CIEF experiment using the set of peptide pI markers allows the determination of the pI of a protein or a peptide within an average error of less than 0.1 pH unit. This error is quite acceptable for the characterization of a protein for most purposes. The pI determination can be completed within 1 h without any additional time required for staining and destaining with a sample consumption of 10-100 ng for each analysis. The distribution of large differences between the determined values by CIEF and the reference values was not even, but showed some tendency to accumulate in some proteins (Table 2). Trypsin inhibitor was one of such proteins whose pI value was difficult to precisely determine by CIEF. This may be related to the nature of this protein, in that it becomes focused slowly. To confirm the completion of focusing, one additional run would be necessary with a different focusing time.10 Another possible source of the error is a curvature of a pH profile that undermines the assumption of a linear relation of pH vs detection time between two pI markers. An exceptionally large error was observed for trypsin inhibitor with Pharmalyte 3-10, whose pH profile severely bends at pH 4-5 under the present conditions. Fitting the pH vs detection time plot of pI markers to a single straight line over an entire pH gradient was found to not always be appropripate, although it has been used often in previous publications.9-12 For the pI determination of neutral to acidic proteins, Servalyt 3-7 provided better results with a linear pH profile than Phar-

malyte 2.5-8 under our experimental conditions. When a wide pH range carrier ampholyte is used, Pharmalyte 3-10 was more effective for neutral to basic proteins and Servalyt 3-10 was more successful for neutral to acidic proteins. Since our survey is limited to a small number of proteins, such a preference of the carrier ampholytes relative to the effectiveness at different pH ranges may not be generalized to other proteins. With this type of instrument, having a fixed detection point, a mobilization process of an established pH gradient is indispensable when a neutral, coated capillary is used. This detection scheme compels different samples to be focused for different times within a pH gradient and this can compromise the choice of focusing time, i.e., too short for the basic end and too long for the acidic end. Another problem of this type of detection scheme is its onetime nature, and, thus, to ascertain the completion of focusing, another experiment must be repeated from the beginning. Scanning or imaging detection schemes have been proposed for CIEF.5,6 These allow the time course of the focusing process to be followed and the confirmation of the completion of focusing to be simplified. An instrument for CIEF with imaging UV detector has recently commercialized,20 and such an instrument should further facilitate the precise pI determination of proteins by CIEF. The peptide pI markers are not yet commercially available. Commercial peptide synthesis services should be able to supply these peptides on a several-milligrams scale at a reasonable cost. However, utilizing all 15 of the peptides may be too much of an expenditure for a single laboratory, and in addition, the amount of peptides obtained from such services is too much for this purpose. Hopefully, some companies will undertake the distribution of these peptide pI markers to CIEF users.

(20) Wu, J.; Watson, A. H.; Torres, A. R. Am. Biotechnol. Lab. 1999, (May).

AC000387O

Received for review April 6, 2000. Accepted June 28, 2000.

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