http://www.abbs.info e-mail:[email protected]

ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(4): 423-432                                     CN 31-1300/Q

Protein Isoforms Observed by Ultrahigh Resolution Capillary Isoelectric Focusing-electrospray Ionization Mass Spectrometry

LIU Tao, SHAO Xiao-Xia, ZENG Rong, XIA Qi-Chang*

( Research Centre for Proteomic Analysis, Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology,

Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences,Shanghai 200031, China )

Abstract    On-line coupling of capillary isoelectric focusing (CIEF) to electrospray ionization mass spectrometry (ESI-MS) as a two-dimensional separation/analysis system was employed for high-resolution analysis of the protein isoforms observed during CIEF process. The analytical system was established by using neutral coated long capillary (80 cm), active capillary positioning and sheath-liquid interface. Proteins were separated and resolved in CIEF according to their differences in isoelectric point (pI), and then characterized by ESI-MS. The focused protein zones were eluted to the entrance of MS by combining cathodic mobilization with gravity. The ultrahigh resolution (difference in pI<0.04) of this technique obtained under certain conditions led to the detection of three isoforms in hemoglobin A and in sickle cell hemoglobin (with similar charge distribution and same molecular weight, but their differences in pI ranging from 0.04 to 0.08) and two isoforms of b-lactoglobulin A (difference in pI is 0.6). The isoelectric points, relative amounts, and molecular masses of these isoforms were determined simultaneously by CIEF-ESI-MS.

Key words    capillary isoelectric focusing; electrospray ionization mass spectrometry; protein isoform; hemoglobin variants

Capillary isoelectric focusing (CIEF) is a powerful tool with ultrahigh resolving power and high speed. This technique originally developed by Hjerten and his colleagues[1-3] uses neutrally coated[such as linear polyacrylamide (LPA) and polyvinyl alcohol (PVA) coatings] fused silica capillaries to eliminate electroosmotic flow and reduce protein adsorption to the capillary walls. The capillary is filled with a solution of analytes and ampholytes with or without polymer. The ampholytes build up a pH gradient between acidic and basic solutions. The negatively charged ampholytes migrate toward the anode and the positively charged ampholytes migrate toward the cathode when a focusing voltage is applied and the local pH within the capillary will change continuously until all ampholytes reach their pIs. As amphoteric macromolecules, proteins behave in the same way as ampholyte species; they are focused and concentrated into narrow zones in the pH gradient according to their pI values[4]. After focusing is completed, there will be no movement of proteins in the capillary. Protein bands and the entire pH gradient must be mobilized to the detector by chemical method or by hydraulic method, or a combination of these methods. The pH gradient obtained by chemical mobilization is nonlinear, while that obtained by hydraulic mobilization is linear.

When CIEF is coupled to mass spectrometry (MS), information-rich results can be obtained to facilitate direct protein characterization. The CIEF-MS combination is equivalent to 2-DE, a separation based on pI followed by an orthogonal separation based on mass, but with even higher precision. Separation of pI differences as small as 0.004 pH units by CIEF was reported[5]; MS allows the precise molecular mass determination with an error of ±0.01% and has the potential of obtaining structural information when it is operated in collision induced dissociation[6-8] (CID) or electron capture dissociation[9] (ECD) mode. The coupling of CIEF to electrospray ionization (ESI) MS has been accomplished by using sheath-liquid interface[10-19] and liquid-junction interface[20-23]. CIEF can also be off-line coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) by using sheath flow fraction collection system[24, 25]. In addition, the microfabricated IEF device for direct ESI-MS was also reported recently[26].

CIEF-ESI-MS is extremely useful for the analysis of mixtures of proteins with very close pI values. Hemoglobin A and hemoglobin F, with a pI difference of 0.05 pH units, were almost baseline resolved and characterized in CIEF-ESI-MS[11]. CIEF-ESI-MS has been used to analyze glycoform distribution[12], protein phosphorylation[13], and protein refolding intermediates[14], and the screening for high-affinity ligands of the Src SH2 domain[15]. One of the most ambitious and promising applications of this technique is the two-dimensional analysis of the whole cellular proteins by CIEF-ESI-MS[16]. In addition, the use of isotope labeling of the cell culture media can not only improve the mass measurement accuracy but also provide a means for the quantitative proteome-wide measurement of protein expres-sion[18].

In this study, ultrahigh-resolution CIEF-ESI-MS analytical system was established using long LPA-coated capillary and sheath-liquid interface with active capillary positioning[19]. The protein isoforms (proteins with same mass, but different pI) of several standard proteins including cytochrome c, myoglobin and b-lactoglobulin A, and hemoglobin variants were analyzed.

1  Materials and Methods

1.1  Materials

All chemicals used were of analytical grade if not mentioned otherwise. Formic acid, glacial acetic acid, and standard proteins including myoglobin (horse heart), carbonic anhydrase II (bovine erythrocytes, pI 5.9), b-lactoglobulin A (bovine milk), human hemoglobin-A (HbA) and human sickle cell hemoglobin (HbS) were purchased from Sigma (St. Louis, MO, USA); cytochrome c was obtained from Pierce (Rockford, IL, USA); carrier ampholytes Pharmalyte 3.5-10 and Pharmalyte 5-8 were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden); HPLC grade methanol (MeOH) was obtained from Fisher (Fair Lawn, NJ, USA), and all water was distilled and deionized (18 W) using a Milli-Q system from Millipore (Bedford, MA, USA). HbA and HbS were desalted using a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 0.6% acetic acid and freeze-dried, other standard proteins were used as received.

1.2  Methods

CIEF-ESI-MS  Beckman P/ACE system 5500 (Beckman, Fullerton, CA, USA) was coupled on-line to a Finnigan MAT LCQ electrospray ion trap mass spectrometer (San Jose, CA, USA) via a sheath-liquid interface supplied by Finnigan. For the combination of CIEF with ESI-MS, a BioCAPTM linear polyacrylamide (LPA) coated capillary (80 cm × 50 mm I.D., 360 mm O.D., Bio-Rad, Hercules, CA, USA) assembled in Beckman eCAPTM capillary cartridge was mounted within the electrospray probe. A micrometer head attached to the ESI probe allowed fine positioning of the capillary. The polyimine coating at the outlet side of the capillary was burned off 2-3 mm and washed off with ethanol to ensure the electric contact. The LCQ was tuned and calibrated by infusing an aqueous solution of angiotensin (Sigma) and UltramarkTM (Finnigan), respectively.

On-line coupling of CIEF to ESI-MS was established by using a strategy of “active capillary positioning” modified from the method described by Kirby et al.[19]. Before CIEF, the whole CE apparatus was placed on a height-adjustable platform and care was taken to ensure that the CE buffer reservoir and the electrospray end of the capillary were at the same level to avoid the influence of gravity flow. To start a run, the focusing catholyte[1% NH4OH (v/v)] used as liquid sheath was delivered at a flow rate of 2 mL/min by syringe pump on LCQ, and the gas sheath was set to 20 arbitrary units to prevent liquid accumulation on the electrospray needle. Next, the capillary was filled with a mixture of sample and ampholyte[≈0.1 mg of protein per mL of 1% Pharmalyte in H2O (w/v)], and the inlet was placed in a buffer reservoir containing focusing anolyte[1%HAc (v/v)]. The outlet was retracted 1.5 mm inside the liquid sheath tube with the aid of micrometer to form a microreservoir at the end of the tube. The tube was flushed with focusing catholyte for 2 min to remove any residual sample and ampholyte in the microreservoir. The gas sheath was decreased to 7 arbitrary units to allow a standing drop of focusing catholyte to form and stabilize on the end of the liquid sheath tube, while the liquid sheath was still delivered at the same flow rate. A constant focusing voltage of +30 kV (375 V/cm) was then applied until the focusing was complete after about 40 min, as shown by a minimum CE current of 1.0 mA. After switching off the voltage, the capillary tip was extruded 1 mm outside the liquid sheath tube with the aid of micrometer. Then, the liquid sheath was quickly replaced by mobilization catholyte (0.25 / 75 / 25, HAc / MeOH / H2O, v/v/v), and the inlet was placed in another reservoir containing mobilization anolyte[0.5% HAc(v/v)]. To combine gravity with cathodic mobilizations, the inlet reservoir was raised 8 cm above the electrospray needle. Finally, the gas sheath was shut off, an ESI voltage of +4.25 kV was applied to the electrospray tip, and a voltage of +30 kV was applied immediately to the capillary (effective mobilization voltage gradient was 322 V/cm), and the data collection was begun.

The ion trap was scanned from m/z 1100 to m/z 2000 at a scan rate of 0.33 s/scan and detected in positive ion mode. Other parameters were as follows: electron multiplier voltage, -950 V; heated capillary temperature, 180 ℃; capillary voltage, +24 V; tube lens offset, -5 V.

2  Results

2.1  CIEF-ESI-MS analysis of model protein mixtures

A mixture of model proteins, cytochrome c, myoglobin, and b-lactoglobulin A was analyzed by ultrahigh resolution CIEF-ESI-MS. The mixture contains 1% (w/v) Pharmalyte 3.5-10, and the following proteins: 8.0 mmol/L cytochrome c; 1.5 mmol/L myoglobin; 8.0 mmol/L b-lactoglobulin A. The protein mixture was focused in an 80 cm long LPA-coated capillary for about 40 min under a constant voltage of 30 kV. The current decreased from 4.5 mA at the beginning to 1.2 mA when the focusing was finished. Figure 1 shows the base peak of CIEF-ESI-MS analysis of the protein mixture. It can be seen that all three proteins were well focused into narrow bands and all eluted within one minute, thus allowing the resolving of proteins with very close pI values. Interestingly, it was observed that each of these three proteins contained two isoforms with almost the same molecular masses, even with longer time of focusing (Data not shown). The isoforms of myoglobin with pI values of 7.2 and 6.8 are well known and are often used as the IEF standard, while cytochrome c can only be detected as a single band with a pI value of 9.6. Yang et al[23] reported that two isoforms of cytochrome c could be found in CIEF-ESI-MS by using the highly efficient microdialysis junction interface, but only one kind of protein could be detected when coaxial liquid sheath interface was used. In our experiment, two cytochrome c isoforms were well resolved although a common sheath liquid interface was used; the increased resolution might be due to the use of long capillary, in which a narrower pH gradient could be established than that in short capillaries using the same ampholyte mixtures. These results indicate that the increased resolution of analytical method may contribute to the detection of protein isoforms separated in IEF process. Besides cytochrome c and myoglobin, a minor isoform of b-lactoglobulin A with very low abundance could also be detected by sensitive CIEF-ESI-MS. The full mass spectra of these isoforms are shown in Figure 2. It can be seen that the charge distribution patterns (and hence the molecular masses) of these protein isoforms are quite similar but their migration times (and hence pIs) are different.

Fig.1  CIEF-ESI-MS base peak of a mixture of cytochrome c (8.0 mmol/L), myoglobin (1.5 mmol/L), and b-lactoglobulin A (8.0 mmol/L)

Remark:  Capillary, 80 cm total length, 50 mm I.D. and 360 mm O.D.; applied voltages, 30 kV for focusing and mobilization, 4.25 kV for electrospray; sheath liquid, acetic acid/methanol/water (0.25/75/25), 2 mL/min; mass scan, m/z 1 100-2 000 at 0.33 s/scan. The inlet reservoir is raised 8 cm above the electrospray needle for the introduction of gravity flow.

Fig.2  Summed mass spectra from the labeled peaks in the base peak in Fig.1

(A1) cytochrome c 1, (A2) cytochrome c 2; (B1) myoglobin 1, (B2) myoglobin 2; (C1) b-lactoglobulin A 1, (C2) b-lactoglobulin A 2.

In cathodic mobilization, the acetate ions in the sheath liquid compete with the hydroxide ions for electromigration into the capillary. Because the pH gradient drifts downward, the focused proteins become positively charged and migrate toward the cathodic end (entrance of MS). Therefore, the proteins previously focused at the cathodic end (basic proteins) migrate ahead of those previously focused at the anodic end (acidic proteins). In addition, the rate of progressive pH change depends on the amount of anion moving into the capillary, the mobility of the anion, and the buffering capacity of carrier ampholytes. As a result, basic proteins are more efficiently mobilized toward the cathodic end (hence exhibit less zone broadening) than acidic proteins at the far end of the capillary[23]. When the gravity flow was combined with cathodic mobilization, poor linear correlation between the migration times of standard proteins and their pI values was observed. Cytochrome c has a pI of 9.6, myoglobin has two pIs of 7.2 and 6.8, and b-lactoglobulin A has a pI of 5.1 at pH range 5-10[Fig. 3(A)]. However, much better linear correlation between migration times and pI values could be obtained in narrow pH range[Fig.3(B),3(C)]. Thus using the strategy of performing linear correlation in narrow pH ranges separately, the pI values of basic and acid proteins can all be determined. The molecular mass, relative abundance and pI value of these protein isoforms determined by CIEF-ESI-MS are summarized in Table 1.

Fig.3  Calibration curves of migration time versus protein pI in CIEF-ESI-MS in different pH range

Another protein mixture containing cytochrome c, myoglobin and carbonic anhydrase II (pI=5.9) was also analyzed by this method (Fig. 4). Again, excellent resolution and a linear correlation coefficient of 0.9975 in pH 5.5-7.5 were obtained. The pI values of two small amphoteric molecules (M1 and M2) contaminating carbonic anhydrase II sample could be accurately assigned as 6.15 and 5.55. Carbonic anhydrase II was efficiently ionized in CIEF-ESI-MS, +15-+26 positively charged ions were detected in m/z 1 100-2 000 (data not shown). Interestingly, only one form of carbonic anhydrase II was found by CIEF-ESI-MS with slight zone broadening after a long period of mobilization.

Fig.4  CIEF-ESI-MS base peak of a mixture of cytochrome c (8.0 mmol/L), myoglobin (2.5 mmol/L), and carbonic anhydrase II (3.0 mmol/L)

Other conditions are the same as in Fig.1.

2.2  CIEF-ESI-MS analysis of hemoglobin variants

Hemoglobin is well known of the existence of a large number of variants, some variants can result in the malfunctioning of the red cells, such as sickle cell hemoglobin. Studies on hemoglobin variants are of great significance not only in basic research but also in practice. The combined use of pI and mass data can greatly facilitate the identification of different variants. For example, although the mass shift between Hb C variant and Hb A is only 0.94 amu, which is not so easy to differentiate them singly by ESI-MS, but the pI shift of 0.5 pH unit between them can be easily detected by CIEF. The pI values of the most common four hemoglobin variants were determined previously (Hb C, 7.45; Hb S, 7.20; Hb F, 7.0; Hb A, 6.95).

In this study, a mixture of Hb S and Hb A was analyzed by high-resolution CIEF-ESI-MS. Figure 5 shows the base peaks of the separations using Pharmalyte 3.5-10 and Pharmalyte 5-8. There were only two proteins in the mixture, but the base peaks of the separations were more complicated. However, when selected ion monitoring (SIM) was used to locate Hb S and Hb A (+12 charged ions with m/z values of 1320.9 and 1323.4 were chosen for themonitoring of Hb S and Hb A, respectively), it was found that the complexity of the base peak of separation was due to the presence of 6 isoforms of hemoglobins with very close pI values (3 isoforms of each variant). Moreover, the three isoforms of the same hemoglobin (nominate as Hb 1, Hb 2 and Hb 3 according to their migration times) was baseline resolved. Closer inspection revealed that the three isoforms of Hb S had the same charge distribution pattern and molecular mass, the same results were also observed in isoforms of Hb A (Fig. 6). Two isoforms of Hb S (Hb S2 and Hb S3) were contaminated by two isoforms of Hb A (Hb A1 and Hb A2), because their pI values were too close. When ampholytes with narrow pH range was used in CIEF-ESI-MS, the resolution of different isoforms could be further improved[Fig. 5（B）].

Fig.5  CIEF-ESI-MS base peak (top panel) and SIM (middle and bottom panels) of a mixture of hemoglobin S (3.0 mmol/L) and hemoglobin A (3.0 mmol/L) using (A) Pharmalyte 3.5-10 and (B) Pharmalyte 5-8

Fig.6  Summed mass spectra from the labeled peaks in Fig. 5(A)

(A) Hb S1; (B) Hb A3. The insets show the mass of hemoglobin a-chain (labeling with A), bs-chain (labeling with Bs), and (ba-chain (labeling with Ba)

In order to determine the pI values of these hemoglobin isoforms, cytochrome c and myoglobin were added to hemoglobin variants and the mixture was separated again by CIEF-ESI-MS (Fig.7). A correlation coefficient of 0.9967 was obtained in the pH 6.5-10 using cytochrome c and myoglobin as standards. Using SIM to find the exact migration times of each hemoglobin isoforms, their pI values were precisely determined. The detection results of these hemoglobin isoforms are summarized in Table 2. It can be seen that, Hb S2 and Hb A1 have very close pI values (7.18 and 7.17); Hb S3 and Hb A2 also have very close pI values (7.13 and 7.11), thus the corresponding isoforms cannot be well resolved. The difference in pI values between Hb A2 and Hb A3 is only 0.04 pH unit, yet they could be separated completely.

Fig.7  CIEF-ESI-MS base peak (top panel) and SIM (middle and bottom panels) of a mixture of hemoglobin S (3.0 mmol/L), hemoglobin A (3.0 mmol/L), and model proteins cytochrome c (4.0 mmol/L) and myoglobin (0.8 mmol/L) using Pharmalyte 3.5-10

Other conditions are the same as in Fig. 1.

The mechanism of the formation of such protein isoforms in CIEF process is not clear, but the possibility that it is formed due to the protein precipitation can be eliminated. For example, the hemoglobin variants have been separated by CIEF-ESI-MS at the concentration ranging from 10-5-10-7 mol/L, but no such isoforms were found[11], yet the protein concentration used in this study is at the range of 10-6-10-7　mol/L. In addition, two isoforms of cytochrome c could be detected when a microdialysis junction interface was used even at a concentration of 10-7 mol/L[23].

3  Discussion

Comparing to other techniques, CIEF-ESI-MS can easily resolve isoforms with tiny pI difference. For example, Hb A2 and Hb A3 with a pI difference of 0.04 pH units can be baseline resolved by this technique. Other advantages of CIEF-ESI-MS include simple sample preparation, high sensitivity, and the possibility of automation. The information-rich data collected in CIEF-ESI-MS can greatly facilitate the identification of proteins. CIEF-ESI-MS combination also provides another orthogonal separation possibility in addition to capillary zone electrophoresis-mass spectrometry (CZE-MS), liquid chromatography-mass spectrometry (LC-MS), and 2-DE techniques. For example, the protein isoforms found in this study cannot be detected by CZE-MS or LC-MS. The complementation of these techniques not only can make the characterization of proteins more reliable but also may contribute to the discovering of interesting molecular processes.

Another interesting phenomenon found in this study is: some proteins, such as cytochrome c, myoglobin, and b-lactoglobulin A, have two isoforms with identical molecular masses, in the case of hemoglobin, even three isoforms were found for each variant; while other proteins (e.g. carbonic anhy-drase II) do not have such isoforms. Although theories of structural biology and dynamics may help to explain the reason why such protein isoforms can form after IEF, deep-going investigations in different fields, such as protein behavior in IEF, structural study of such isoforms, are still need to perform to further explore the exact mechanism.

Acknowledgements    The authors thank Prof. WANG Ke-Yi in our institute and Dr. ZHU Ming-De in Bio-Rad laboratories for the helpful discussion.

References

1  Hjerten S, Zhu MD. Adaptation of the equipment for high-performance electrophoresis to isoelectric focusing. J Chromatogr, 1985, 346: 265-270

2  Hjerten S, Liao JL, Yao KQ. Theoretical and experimental study of   high-performance electrophoretic mobilization of isoelectrically focused protein zones. J Chromatogr, 1987, 387: 127-138

3  Kilar F, Hjerten S. Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries. Electrophoresis, 1989, 10: 23-29

4  Mazzeo JR, Krull IS. Capillary isoelectric focusing of proteins in uncoated fused-silica capillaries using polymeric additives. Anal Chem, 1991, 63: 2852-2857

5  Shen Y, Xiang F, Veenstra TD, Fung EN, Smith RD. High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms. Anal  Chem, 1999, 71: 5348-5353

6  Flora JW, Hannis JC, Muddiman DC. High-mass accuracy of product ions produced by SORI-CID using a dual electrospray ionization source coupled with FTICR mass spectrometry. Anal  Chem, 2001, 73: 1247-1251

7  Cheng Y, Hercules DM. Studies of pesticides by collision-induced dissociation, postsource-decay, matrix-assisted laser desorption/ionization time of flight mass spectrometry. J Am Soc Mass Spectrom, 2001, 12: 590-598

8  Chang Y, Abliz Z, Li LJ, Fang QC, Takayama M. Study on fragmentation behavior of 5/7/6-type taxoids by tandem mass spectrometry. J Mass Spectrom, 2000, 35: 1207-1214

9  Zubarev RA, Horn DM, Fridriksson EK, Kelleher NL, Kruger NA, Lewis MA, Carpenter BK et al. Electron capture dissociation for structural characterization of multiply charged protein cations. Anal Chem, 2000, 72: 563-573

10  Tang Q, Harrata AK, Lee CS. Capillary isoelectric focusing-electrospray mass spectrometry for protein analysis. Anal Chem, 1995, 67: 3515-3519

11  Tang Q, Harrata AK, Lee CS. High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis. Anal  Chem, 1996, 68: 2482-2487

12  Yang L, Tang Q, Harrata AK, Lee CS. Capillary isoelectric focusing-electrospray ionization mass spectrometry for transferrin glycoforms analysis. Anal  Biochem, 1996, 243: 140-149

13  Wei J, Yang L, Harrata AK, Lee CS. High resolution analysis of protein phosphorylation using capillary isoelectric focusing-electrospray ionization-mass spectrometry. Electrophoresis, 1998, 19: 2356-2360

14  Jensen PK, Harrata AK, Lee CS. Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry. Anal Chem, 1998, 70: 2044-2049

15  Lyubarskaya YV, Carr SA, Dunnington D, Prichett WP, Fisher SM, Appelbaum ER, Jones CS et al. Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry. Anal Chem, 1998, 70: 4761-4770

16  Tang W, Harrata AK, Lee CS. Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal Chem, 1997, 69: 3177-3182

17  Zhang CX, Xiang F, Pasa-Tolic L, Anderson GA, Veenstra TD, Smith RD. Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry. Anal Chem, 2000, 72: 1462-1468

18  Jensen PK, Pasa-Tolic L, Peden KK, Martinovic S, Lipton MS, Anderson GA, Tolic N et al. Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures. Electrophoresis,  2000, 21: 1372-1380

19  Kirby DP, Thorne JM, Gotzinger WK, Karger BL. A CE/ESI-MS interface for stable, low-flow operation. Anal  Chem, 1996, 68: 4451-4457

20  Zhang R, Hjerten S. A micromethod for concentration and desalting utilizing a hollow fiber, with special reference to capillary electrophoresis. Anal Chem, 1997, 69: 1585-1592

21  Severs JC,Smith RD.Characterization of the microdialysis junction interface for capillary electrophoresis/microelectrospray ionization mass spectrometry. Anal Chem, 1997, 69: 2154-2158

22  Lamoree MH, Tjaden UR, van der Greef J. On-line coupling of micellar electrokinetic chromatography to electrospray mass spectrometry. J Chromatogr A, 1995, 712: 219-225

23  Yang L, Lee CS, Hofstadler SA, Pasa-Tolic L, Smith RD. Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization. Anal  Chem, 1998, 70: 3235-3241

24  Foret F, Muller O, Thorne J, Gotzinger W, Karger BL. Analysis of protein fractions by micropreparative capillary isoelectric focusing and matrix-assisted laser desorption time-of-flight mass spectrometry. J Chromatogr A, 1995, 716: 157-166

25  Minarik M, Foret F, Karger BL. Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing. Electrophoresis, 2000, 21: 247-254

26  Wen J, Lin Y, Xiang F, Matson DW, Udseth HR, Smith RD. Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry. Electrophoresis, 2000, 21: 191-197

Received: December 6, 2001    Accepted: January 30, 2002

The work was supported by grants from the Special Funds for Major State  Basic Research Project (973) of China (No.2001CB5102), and by State 863 High-Technology R&D Project of China  (No.2001AA233031)

*Corresponding author: Tel, 86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]