Anal. Chem. 1997, 69, 3177-3182
Two-Dimensional Analysis of Recombinant E. coli Proteins Using Capillary Isoelectric Focusing Electrospray Ionization Mass Spectrometry Qing Tang, A. Kamel Harrata, and Cheng S. Lee*
Department of Chemistry and Ames Laboratory, USDOE, Iowa State University, Ames, Iowa 50011
On-line combination of capillary isoelectric focusing with electrospray ionization mass spectrometry is applied for a two-dimensional analysis of Escherichia coli proteins. The proteins are focused and cathodically mobilized in a polyacrylamide coated capillary. At the end of the capillary, various protein zones are analyzed by mass spectrometry coupled through an electrospray interface. Comparisons with silver-stained two-dimensional gel electrophoresis are made with regard to mass determination, resolution, speed, and sensitivity. Direct identification of a recombinant fusion protein of glutathione S-transferase and striped bass growth hormone is achieved without any prior protein isolation procedures. Traditionally, proteins have been characterized by twodimensional gel electrophoresis.1 All the sample proteins are separated first by isoelectric point (pI) and then by size in a twodimensional gel. Despite the selectivity and sensitivity of twodimensional gel electrophoresis, this technique as practiced today is the collection of manually intensive procedures. Casting of gels, application of samples, running of gels, and staining of gels are time-consuming tasks, prone to irreproducibility and poor quantitative accuracy. In capillary isoelectric focusing (CIEF), the fused silica capillary contains not only ampholytes but also proteins.2-4 When an electric potential is applied, the negatively charged acidic ampholytes migrate toward the anode and decrease the pH at the anodic section, while the positively charged basic ampholytes migrate toward the cathode and increase the pH at the cathodic section. These pH changes will continue until each ampholyte species reaches its pI, where it will then concentrate. Because each ampholyte has its own buffering capacity, a virtually continuous pH gradient forms in the capillary. Protein analytes as amphoteric macromolecules also focus at their pI values in narrow zones in the same way as the individual ampholytes. The focusing effect of CIEF permits analysis of dilute protein samples with a typical concentration factor of 50-100 times. Recent advances in electrospray ionization mass spectrometry (ESIMS) facilitate the formation of multiply-charged ions from high molecular weight biopolymers and the precise mass determination of (0.02% for proteins up to 80 kDa.5-8 In ESIMS, a spray of charged droplets is formed under the influence of an (1) Creighton, T. E. Protein Structure: A Practical Approach; IRL Press: New York, 1990; Chapter 3. (2) Hjerten, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265-270. (3) Hjerten, S.; Liao, J. L.; Yao, J. J. Chromatogr. 1987, 387, 127-138. (4) Kilar, F.; Hjerten, S. Electrophoresis 1989, 10, 23-29. (5) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671-4675. S0003-2700(97)00015-2 CCC: $14.00
© 1997 American Chemical Society
electric potential applied at the end of the capillary. Upon continuous solvent evaporation and droplet disintegration, the highly charged droplets produce gas phase ions by either ion emission or complete solvent evaporation. The multiply-charged phenomenon of ESIMS allows the analysis of large biomolecules (>100 kDa) using mass analyzers with limited m/z range. The objective of this study is to combine the strengths of both CIEF in the ease and speed of electrophoretic separation and ESIMS in the accuracy of mass determination for the twodimensional analysis of Escherichia coli proteins. The proteins are focused and mobilized in a polyacrylamide-coated capillary. At the end of CIEF capillary, the mobilized protein zones are analyzed by ESIMS using a coaxial sheath flow configuration. The use of sheath flow establishes the electrical connection at the CIEF capillary terminus, which serves to define the electric field along the CIEF capillary and apply an electric voltage for electrospray ionization.9 The integration of CIEF with ESIMS holds the promise of putting two-dimensional gel electrophoresis on the same instrumental footing as high-performance liquid chromatography. As demonstrated in this study, CIEF-ESIMS clearly exhibits superior resolving power, speed, and sensitivity for protein characterization. Direct identification of a recombinant fusion protein of glutathione S-transferase (GST) and striped bass growth hormone (sbGH) expressed in E. coli10 is achieved using CIEF-ESIMS without any prior protein isolation and purification procedures. EXPERIMENTAL SECTION Recombinant E. coli Proteins. E. coli cells overexpressing a fusion protein of GST and sbGH were kindly provided by Dr. Antonio R. Moreira at the University of Maryland Baltimore County. The cells were suspended in a buffer which consisted of 10 mM Tris-HCl (pH 7.0), 5 mM magnesium chloride, 0.1 mM dithiothreitol, and 10% glycerol. The cells were disrupted by sonication for the release of cellular proteins.11 After sonication, DNase was added with a final concentration of 50 µg/mL for the digestion and removal of nucleic acids. The cellular proteins were collected in the supernatant by centrifugation at 2000g for 10 min. (6) Fenn, J. B.; Mann, M.; Meng, C. W.; Wong, S. F. Mass Spectrom. Rev. 1990, 9, 37-70. (7) Ikonomou, M. G.; Blades, A.; Kebarle, T. P. Anal. Chem. 1991, 63, 19891998. (8) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (9) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574A-584A. (10) Cheng, C. M.; Lin, C. M.; Shamblott, M.; Gonzalez-Villasenor, L. I.; Powers, D. A.; Woods, C.; Chen, T. T. Mol. Cell. Endocrinol. 1995, 108, 75-85. (11) Schleif, R. F.; Wensink, P. C. Practical Methods in Molecular Biology; Springer-Verlag: New York, 1981; Chapter 1.
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The protein solution was then desalted using a regenerated cellulose membrane (Millipore, Bedford, MA) with a 5000 molecular weight cutoff. The total protein concentration of the resulting solution determined by the Bradford method (Bio-Rad kit, Richmond, CA) was around 12 mg/mL. Capillary Isoelectric Focusing: UV Measurement. The CIEF apparatus was constructed in-house using a CZE 1000R highvoltage (HV) power supply (Spellman High-Voltage Electronics, Plainview, NY). Fused silica capillaries, 50-µm i.d. and 192-µm o.d. (Polymicro Technologies, Phoenix, AZ), were coated internally with linear polyacrylamide for the elimination of electroosmotic flow.4 The protein zones were monitored by a UV detector (Linear Instruments, Reno, NV) at 280 nm. For the creation of a UV detection window, a 2-mm section of polyimide coating on the exterior surface of the capillary was removed using hot sulfuric acid. A 30-cm-long capillary (27 cm to UV detector) was rinsed with deionized water and then filled with a solution containing carrier ampholytes and E. coli proteins. A total of 7 µg (12 mg/mL × 0.59 µL of capillary volume) of E. coli proteins was loaded in the CIEF capillary. Focusing was performed at a 10-kV constant voltage for 7 min with the use of 20 mM phosphoric acid and 20 mM sodium hydroxide as the anolyte and the catholyte, respectively. Cathodic mobilization was initiated by replacing the sodium hydroxide catholyte with a solution containing methanol/water/ acetic acid in a volume ratio of 50:49:1 at pH 2.6. To induce the gravity mobilization, the inlet reservoir was raised 8 cm above the outlet reservoir. A constant voltage of 10 kV was again applied during the mobilization. Carrier ampholytes of pharmalyte 3-10 and pharmalyte 5-8 were obtained from Pharmacia (Uppsala, Sweden). The concentration of ampholyte mixture containing pharmalyte 5-8 and pharmalyte 3-10 at a ratio of 3:1 was varied between 2% and 0.5% in the CIEF capillary. A mixture of standard proteins including glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle, 35 700 Da, pI ) 8.3 and 8.5), myoglobin (horse heart, 16 950 Da, pI ) 6.8 and 7.2), conalbumin (hen egg, 77 500 Da, pI ) 6.6, 6.3, and 6.0), carbonic anhydrase II (bovine serum, 29 024 Da, pI ) 5.9), albumin (bovine serum, 66 200 Da, pI ) 5.6, 5.5, and 5.4), and trypsin inhibitor (soybean, 20 091 Da, pI ) 4.5) was purchased from Sigma (St. Louis, MO). All chemicals, including acetic acid, dithiothreitol, glycerol, magnesium chloride, methanol, phosphoric acid, sodium hydroxide, and Tris-HCl, were purchased from Fisher (Fair Lawn, NJ). All solutions were filtered through a 1-µm filter (Whatman, Maidstone, England). Mass Spectrometer and Electrospray Interface. The mass spectrometer was a Finnigan MAT TSQ 700 (San Jose, CA) triple quadrupole equipped with an electrospray ionization source. The Finnigan MAT electrospray adapter kit, containing both gas and liquid sheath tubes, was used to couple CIEF with ESIMS without any modifications. The electrospray needle was maintained at 5 kV for all CIEF-ESIMS measurements. The first quadrupole was used for the mass scanning of protein ions, while the second and third quadrupoles were operated in the radio frequency-only mode. The electron multiplier was set at 1.4 kV, with the conversion dynode at -15 kV. Tuning and calibration of the mass spectrometer were established by using an acetic acid solution (methanol/ water/acetic acid, 50:49:1 v/v/v) containing myoglobin and a small peptide of methionine-arginine-phenylalanine-alanine. 3178 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
Figure 1. CIEF-UV separation of recombinant E. coli proteins in the presence of various ampholyte concentrations. Capillary, 30-cm total length, 50-µm i.d., and 192-µm o.d.; length of capillary to detector, 27 cm; voltage, 10 kV for focusing and mobilization; UV detection at 280 nm. Ampholyte concentrations: (A) 0.5% pharmalyte, (B) 1% pharmalyte, (C) 2% pharmalyte.
Capillary Isoelectric Focusing Electrospray Ionization Mass Spectrometry. For the combination of CIEF with ESIMS, a 30-cm-long CIEF capillary was mounted within the electrospray probe. The outlet reservoir, containing 20 mM sodium hydroxide as the catholyte, was located inside the electrospray housing during the focusing step. The inlet reservoir, containing 20 mM phosphoric acid as the anolyte, was kept at the same height as the outlet reservoir. The capillary dimensions and applied focusing voltage were the same as in the CIEF-UV measurements. Once the focusing was completed, the electric potential was turned off, and the outlet reservoir was removed. The capillary tip was fixed about 0.5 mm outside the electrospray needle. The sheath liquid consisted of 50% methanol, 49% water, and 1% acetic acid (v/v/v) and was delivered at a flow rate of 5 µL/min with the use of a Harvard Apparatus 22 syringe pump (South Natick, MA). During the mobilization step, two high voltage (HV) power supplies (Spellman) were used for delivering the electric potentials of 15 and 5 kV at the inlet electrode and the electrospray needle, respectively. Because most HV power supplies are not designed to operate as current sinks, a resistor ladder, parallel with the HV electrode connecting with the electrospray needle, was incorporated. Detailed configuration of CIEF-ESIMS, including sheath liquid and electrical connections, is described elsewhere.13,14 To combine gravity with cathodic mobilizations, the inlet reservoir was raised 8 cm above the electrospray needle. No sheath gas was employed during the CIEF-ESIMS measurements. The first quadrupole was scanned from m/z 700 to 2000, with a scan rate of 2.5 s/scan. The deconvoluted mass spectra of protein analytes were obtained using the LC/MS BioToolBox analysis software from Perkin-Elmer (Foster City, CA). The mass deconvolution was performed for the scan range of m/z 7002000 and was based on an iterative process. The deconvoluted mass approached the real solution more closely as more iterations were performed. The artifact peaks, which might be present initially due to the incorporation of minor noise peaks in deconvolution, decreased and finally disappeared as the result of the iterative process. More than 50 iterations were executed for the deconvolution of each mass spectrum.
Figure 2. CIEF-ESIMS reconstructed ion electropherogram of E. coli proteins. Capillary, 30-cm total length, 50-µm i.d., and 192-µm o.d.; applied voltages, 10 kV for focusing and mobilization, 5 kV for electrospray; sheath liquid, methanol/water/acetic acid (50:49:1 v/v/v) at pH 2.6, 5 µL/min; mass scan, m/z 700-2000 at 2.5 s/scan; 7 µg of total protein was loaded in the CIEF capillary.
Two-Dimensional Gel Electrophoresis. The procedures for performing two-dimensional gel electrophoresis were described elsewhere.12 For the first dimension, 12.5 µL of cell extract (approximately 150 µg of total protein) was mixed with 12.5 µL of sample buffer and applied to the Bio-Rad gel tube. The sample buffer consisted of 9.5 M urea, 2% Triton X-100, 5% β-mercaptoethanol, 1.5% pharmalyte 5-8, and 0.5% pharmalyte 3-10. Isoelectric focusing was performed by applying 300 V for 10 min, followed by 500 V for 8 h. For the second dimension, the tube gel was equilibrated for 20 min in the sodium dodecyl sulfate (SDS) equilibration buffer (62.5 mM Tris-HCl, pH 6.8, 2.3% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue) and was gently squeezed onto a separating gel (16.5% total acrylamide concentration). The separation was carried out in a Bio-Rad mini 2-D electrophoresis cell by applying 200 V for 45 min. SDS gel was stained with silver immediately following electrophoresis. The stained gel was dried and scanned by a Sharp JX-320 scanner (Mahwah, NJ). RESULTS AND DISCUSSION Recombinant E. coli cells were disrupted by sonication for the release of cellular proteins to be analyzed by CIEF-ESIMS and two-dimensional gel electrophoresis. To investigate the effect of carrier ampholyte concentration on E. coli protein separation in CIEF, the concentration of an ampholyte mixture containing pharmalyte 5-8 and pharmalyte 3-10 at a ratio of 3:1 was varied between 2% and 0.5% in the CIEF capillary. To prevent the ampholytes and proteins from migrating into the inlet and outlet reservoirs by either diffusion or gradient drift,2 the solutions of 20 mM phosphoric acid and 20 mM sodium hydroxide were used as the anolyte and the catholyte, respectively. (12) O’Farrell, P. H. J. Biol. Chem. 1974, 250, 4007-4021.
When the focusing step was completed, focused protein zones were mobilized by combining cathodic mobilization with a gravityinduced hydrodynamic flow. The cathodic mobilization was initiated by replacing the sodium hydroxide catholyte with a solution containing methanol/water/acetic acid in a volume ratio of 50:49:1 at pH 2.6. The catholyte used in cathodic mobilization was the same as that used in the sheath liquid later employed in the electrospray interface for the CIEF-ESIMS measurements. The migration time of protein zones increased with increasing ampholyte concentration (see Figure 1). The increase in solution viscosity with increasing pharmalyte concentration accounted for the longer migration time. In comparison to protein separation at 2% pharmalyte, an approximately 22% loss in resolution was measured in CIEF with 0.5% pharmalyte. The presence of carrier ampholyte in ESI not only caused a marked reduction in the protein ion intensity but also resulted in a decrease of the net charge of protein ions in the mass spectra compared to the average charge state measured in the absence of carrier ampholyte.13,14 Thus, the ampholyte ions similar to simple electrolyte ions led to higher solution conductivity and contributed to the establishment of the charge excess necessary for the electrospray process.15-17 Furthermore, the possible ionpair formation between the anionic moiety of carrier ampholyte and a positively-charged basic amino acid of protein could be qualitatively accounted for by the findings of Mirza and Chait.18 Ion-pair formation in combination with the subsequent desolvation and dissociation processes resulted in the charge neutralization (13) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1995, 67, 3515-3519. (14) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1996, 68, 2482-2487. (15) Tang, L.; Kebarle, P. Anal. Chem. 1991, 63, 2709-2715. (16) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (17) Wang, G.; Cole, R. B. Anal. Chem. 1994, 66, 3702-3708. (18) Mirza, U. A.; Chait, B. T. Anal. Chem. 1994, 66, 2898-2904.
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Figure 3. Positive ion electrospray mass spectrum taken from the average scans under the peak 14 of Figure 2. Each symbol represents a separate protein ionization envelope. The inset shows a deconvoluted mass spectrum of protein analytes.
Figure 4. Distribution of pI and molecular weight of E. coli proteins measured by CIEF-ESIMS. Standard proteins used for the correlation of migration time with the corresponding pI value are represented by open circles.
effect and the shift in the charge distribution of protein mass spectra. Considering the effects of ampholyte molecules on the CIEF protein separation and the protein electrospray ionization mass spectra, a solution containing 0.5% carrier ampholyte (pharmalyte 5-8 and pharmalyte 3-10 at a ratio of 3:1) and E. coli proteins was used for all CIEF-ESIMS measurements. The blending of pharmalyte not only gave an effective separation range between pH 3.5 and 9.5, but also enhanced separation resolution and pH gradient in the range of 5-8. In the CIEF-ESIMS experiments, the CIEF separation conditions including capillary dimensions and electric field strengths during the focusing and mobilization steps were identical to those employed in the CIEF-UV studies. However, the longer migration distance from the focused regions to the end of the capillary in CIEF-ESIMS accounted for the increase in the migration time (see Figure 2). In order to maintain electroneutrality, the electric charge transported by the ions exiting one end of the CIEF capillary must 3180 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
Figure 5. Silver-stained two-dimensional gel electrophoresis of E. coli proteins; 150 µg of total protein was applied to the gel. Size and pI ranges are indicated on the vertical and horizontal axes, respectively.
be substituted by a charge carried by ions of the same sign entering the opposite end of the capillary. In the absence of electroosmotic flow, the sheath liquid counterions used in the electrospray interface migrated into the CIEF capillary, replacing the original counterions of the background electrolyte. The moving ionic boundary led to delays or inversions in migration order and resulted in loss of separation resolution in CIEFESIMS.14 In this study, the formation of a moving ionic boundary from the sheath liquid was minimized by introducing a gravityinduced flow, together with cathodic mobilization of focused protein zones in the CIEF capillary. During the mobilization step, the inlet reservoir was raised 8 cm above the electrospray needle. In comparison to the CIEF-UV studies (see Figure 1), the scan rate of the quadrupole mass spectrometer might be insufficient to truly reflect high separation efficiency and resolution of CIEF and resulted in an apparently poorer resolution of E. coli proteins in the reconstructed ion electropherogram (see Figure 2). The reconstructed ion electropherogram reflected the major separation pattern of E. coli proteins as observed in CIEF-UV. However, small differences arose due to the nature of analyte
Figure 6. Positive ion electrospray mass spectrum taken from the average scans under the peak 16 of Figure 2. The mass ions of GSTsbGH are marked with their charge numbers. The inset shows a deconvoluted mass spectrum of GST-sbGH.
detection by UV absorbance versus gas phase ion intensity. Approximately 26 peaks and shoulders were identified for various protein zones with different pI values. External correlation of migration time in CIEF-ESIMS with the corresponding pI value was established using a mixture of standard proteins including glyceraldehyde-3-phosphate dehydrogenase, myoglobin, conalbumin, carbonic anhydrase II, albumin, and trypsin inhibitor. An excellent linear correlation coefficient of 0.992 was obtained with the accuracy of pI determination within (0.1 pH unit. Additionally, a mass determination accuracy of 0.01-0.04% between the expected and measured molecular masses of standard proteins was demonstrated for the precise mass determination. The mass spectrum obtained from the average scans under the peak 14 was illustrated (see Figure 3) and employed for the deconvoluted analysis of protein mass. The migration time of peak 14 corresponded to a pI value of 6.3. Four separate electrospray ionization envelopes with various ion intensities were observed, indicating the coelution of four protein analytes within the peak 14. The inset in Figure 3 presented the deconvoluted mass spectrum for the protein masses of 13 698, 27 378, 28 457, and 28 901 Da. By analyzing all 26 protein zones in the reconstructed ion electropherogram, the distribution of pI and molecular weight of E. coli proteins were presented in a two-dimensional plot (see Figure 4), similar to that obtained by two-dimensional gel electrophoresis. A total of 104 protein molecules was measured with the darkness of short bar for the representation of its relative ion intensity in the mass spectrum. For comparison, a twodimensional gel electrophoresis was employed for the analysis of the same E. coli protein sample using silver staining. The number of identifiable protein spots on a two-dimensional gel (see Figure 5) was around 110, slightly more than that measured by CIEFESIMS. The distribution patterns of E. coli proteins analyzed by CIEF-ESIMS, in general, were consistent with those observed on the two-dimensional gel. The amounts of E. coli proteins loaded in the CIEF capillary and in the two-dimensional gel electrophoresis were 7 and 150
µg, respectively. The difference in protein loading might explain the discrepancy in the numbers of protein molecules measured by CIEF-ESIMS and two-dimensional gel electrophoresis, particularly for the minor cellular proteins in E. coli. Nonlinear calibrations of pI and molecular weight in two-dimensional gel electrophoresis (see Figure 5) were acquired using a mixture of standard proteins. The accuracy of mass determination in ESIMS was around (0.04%, much better than the (5% in SDS polyacrylamide gel electrophoresis.1 Furthermore, it took less than 1 h to complete an entire CIEF-ESIMS analysis, while one whole day was needed to perform two-dimensional gel electrophoresis including protein staining. Additional 6-8 h was needed for the deconvolution of protein mass spectra using the LC/MS BioToolBox analysis software (see Experimental Section). sbGH is a single-chain polypeptide produced by somatotrophs in the anterior portion of the pituitary gland.10 The net charge on a protein is given by19
net charge )
∑n /[(K /[H
+
i
i
] + 1)] -
∑n /[([H
]/Kj + 1)] (1)
+
j
where the molecule has i weakly basic groups and j weakly acidic groups. Kis and Kjs are the ionization constants of basic and acidic moieties, respectively. The assumption is that the ionization constant of a specific moiety is unaffected by its position and neighboring amino acids. Based on the primary structures of sbGH10 and GST,20 the pI and molecular mass of fusion proteins, GST-sbGH, were therefore estimated to be 6.0 and 48 373 Da, respectively. The migration time of peak 16 (see Figure 2) in CIEF-ESIMS corresponded to a pI value of 6.0. By investigating the mass spectrum obtained from the average scans under peak 16 (see (19) Mosher, R. A.; Saville, D. A.; Thormann, W. The Dynamics of Electrophoresis; VCH: New York, 1992; Chapter 7. (20) Smith, D. B.; Davern, K. M.; Board, P. G.; Tiu, W. U.; Garcia, E. G.; Mitchell, G. F. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8703-8707.
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Figure 6), an electrospray ionization envelope corresponding to a deconvoluted mass of 48 394 Da was clearly observed. The accuracy of mass determination was less than 0.04%. GST (pI ) 5.8 and 27 474 Da), which was one of the major proteins expressed by this recombinant E. coli cell line, was clearly identified by CIEF-ESIMS (see Figure 4). A very intense protein region extending between pI ) 5.5 and 5.9, with a molecular mass around 29 000 Da, was also observed in two-dimensional gel electrophoresis (see Figure 5). The observed GST could be the result of both overexpression and endogenous bacterial protein. The overexpression of GST and GST-sbGH might contribute to fewer E. coli proteins being analyzed by CIEF-ESIMS and silverstained two-dimensional gel electrophoresis in this study. Typically, the number of identifiable E. coli protein spots was around 200-300 on a silver-stained two-dimensional gel.1 Additionally, the sample preparation procedure may have led to some loss of E. coli proteins, particularly the membrane-bound proteins. In conclusion, the total E. coli proteins obtained from cell lysis are analyzed by CIEF-ESIMS and compared with silver-stained two-dimensional gel electrophoresis for protein characterization. The recombinant fusion protein of GST-sbGH can be directly identified by CIEF-ESIMS with a mass accuracy of less than 0.04%. The integration of CIEF with ESIMS, as a novel twodimensional bioanalytical methodology, clearly exhibits superior resolving power, speed, and sensitivity and may have practical utility in the biopharmaceutical industry as the means to demonstrate lot-to-lot consistency. However, the detection sensitivity of radiolabeled two-dimensional gel electrophoresis may be about 1-2 orders of magnitude higher than that achievable using CIEFESIMS. (21) Wu, J.; Pawliszyn, J. Anal. Chem. 1995, 67, 2010-2014.
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The practical scope and limitations of CIEF-ESIMS as applied to biological samples include the need to desalt the sample prior to the CIEF separation (see Experimental Section). The presence of high salt concentrations disrupts or destroys the formation of pH gradient during the focusing step. Wu and Pawliszyn21 recently introduced an on-line dialysis system for the removal of salts and the addition of ampholyte molecules. Additionally, the limitations of LC/MS BioToolBox analysis software (see Experimental Section) employed in this study for the deconvolution of protein masses remain to be investigated. Excellent mass determination accuracy of 0.01-0.04% is obtained for a mixture of six standard proteins. However, the upper limit for the number of protein electrospray ionization envelopes which can be reliably and confidently deconvoluted has to be addressed. Finally, the potential ion suppression effect of highly chargeable proteins against other protein analytes coeluted from the CIEF capillary affects the qualitative and quantitative determination of protein molecules in ESIMS. ACKNOWLEDGMENT Support for this work by the Microanalytical Instrumentation Center of the Institute for Physical Research and Technology at Iowa State University is gratefully acknowledged. Q.T. is a recipient of a Pfizer Analytical Chemistry Fellowship. C.S.L. is a National Science Foundation Young Investigator (BCS-9258652).
Received for review January 2, 1997. Accepted June 2, 1997. AC970015O X
Abstract published in Advance ACS Abstracts, July 1, 1997.
