Anal. Chem. 1994,66, 4400-4407
Optimization of Ondine Peptide Mapping by Capillary Zone Electrophoresis Larry Licklider and Werner 0. Kuhr" Depattment of Chemistw, University of California, Riverside, California 9252 1
Avariety of enzyme-catalyzedreactions can be performed in a enzyme-modified fused-silica capillary microreactor which has been integrated with a CZE separation capillary via an open fluid junction. In this report, we describe the utilization of two proteases (trypsin and pepsin) and a peptidase, carboxypeptidase-Y, in the coupled enzymemodified microreactor-CZE system to perform on-line analysis of protein hydrolysates. Several ditrerent proteins, including insulin and a heavily glycosylated protein, al-acid glycoprotein, were digested in the microreactor. Electrophoretic fluid transfer of an aliquot of the total microreactor sample (approximately 50 pmol of denatured glycoprotein) to the separation capillary minimizes loss of the hydrolysate sample and conserves the remainder for iterative injections. Carboxypeptidase-Ymediated hydrolysis of the carboxyl termini of the insulin B-chain was accomplished with on-line CZE analysis of the truncated peptides. Peptide bond cleavages within bovine insulin B-chain were rapidly analyzed in the pepsin microreactor-CZE capillary coupled system, and the electropherogram is compared with a CZE analysis of the free solution pepsin digestion products of the insulin B-chain. Recent developments in the analysis of ultramicroquantities of biomolecules by CZE include on-capillary enzyme-catalyzed reactions to assay enzyme activityl-3 or to produce a protein hydrolysate for peptide mapping! These analyses demonstrate the extremely promising capabilities of capillary zone electrophoresis to accomplish multiple processes in 50 or 75 pm inner diameter capillaries, including, differential migration and mixing of the reactants, variable reaction intervals, and separation and detection of the resulting reactant-product mixture. The principal benefits of these on-capillary manipulations of solutes include the minimal dilution or loss of the reaction products and minimal consumption of the reactants. High overall specificity is provided by the combination of enzyme specificity and a CZE separation of the reaction mixture, as has been shown by CZE analyses of offcapillarytrypsin However, incompatibilities may arise when otherwise distinct processes are joined on the capillary. In an on-capillary protein hydrolysis for peptide mapping, a stop Bao, J. M.; Regnier, F. E. J Chromatugr. 1992,608,217-224. Ada, L. 2.; Whitesides, G. M.J Org. Chem. 1993,58,5508-5512. Xue, 9.;Yeung, E. S. Anal. Chem. 1994,66,1175-1178. Chang, H. T.; Yeung, E. S. Anal. Chem. 1993,65,2947-2951. Cobb, K A; Novotny, M.And. Chem. 1989,61, 2226-2231. Wheat, T. E.; Young, P. M.; Astephen, N. E.]. Liq. Chrumntogr. 1991,14, 987-5396, (7) Amankwa, L. A; Kuhr, W. G. Anal. Chem. 1992,64,1610-1613.
(I) (2) (3) (4) (5) (6)
4400 Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
flow incubation period within the capillary is required to allow numerous hydrolysis products to accumulate before accomplishing their electrophoretic separation. The resolving power of the separation suffers when diffusional broadening of the reaction mixture zone becomes intolerable after lengthy incubation periods, and increasing dilution of the reaction zone with incubation time results in a concomitant reduction in the sensitivity of the oncapillary detection.* Traditionally, relatively slow reaction kinetics for protein hydrolysis generally require long incubation times, which can limit the usefulness of oncapillary proteolytic reactions. Another critical consideration is the suitability of the separation buffer to the enzyme-mediated chemistry. One must also have the capability to mix the enzyme with the chosen substrate (s), maximize conversion of substrate to products, and accomplish a separation of the resulting reaction mixture. These processes are generally optimal under different solution conditions (pH, ionic strength, buffer additives) in the capillary. The success of oncolumn peptide mapping is particularly vulnerable when the optimal pH of the proteolysis reaction differs from the pH at which optimal selectivity is obtained for the separation of the peptides. The goal of this work is to conduct a variety of proteolytic enzymecatalyzed reactions in an enzymemodified fused-silica microreactor which has been integrated with a CZE separation capillary via an open fluid junction. Quantitative transfer of a solute across a solution gap between two capillaries was optimized previously by imaging the transfer of a dye electromigratingacross a wide capillary jun~tion.~ Since the ionic strength of the fluid junction can be lowered 1 order of magnitude below that of the electrophoresis buffer, optimal stacking conditions exist for the transfer of solutes across the fluid junction.1° Fast peptide mapping of a model protein @-casein) was also demonstrated previously using this design." The use of the fluid junction allowed the pH and composition of the microreactor buffer to be independently set without compromising the choice of electrophoresis conditions for high separation efficiency in the CZE capillary. Proteolytic enzymes were immobilized in the microreactor with biotin-avidin technology, which has also been demonstrated to be an effective, general technique in the development of a trypsin-modified capillary ultramicrochemical reactor? In this report, we describe the utilization of two proteases (pepsin and trypsin) and a peptidase, carboxypeptidase-Y (CPY), in the coupled enzymemodified microreactor capillary-CZE system to perform peptide mapping. Reaction times in these microreactors are consistently 30 min or less for complete degradation of linear and (8) Jorgenson, J. W.; Lukacs, IC D. Anal. Chem. 1981,53,1298-1302. (9) Kuhr, W. G.; Licklider, L. J.; Amankwa, L. A Anal. Chem. 1992,65,275282. (10) Burgi, D. S.; Chien, R-L.Anal. Chem. 1991,63,2042-2047. (11) Amankwa, L. A; Kuhr, W. G. Anal. Chem. 1993,65, 2693-2697.
0003-2700/94/0366-4400$04.50/0 Q 1994 American Chemical Society
amorphous structures. Large structurally complex proteins, those of globular form or bearing extensive glycosylation, required optimized microreactor conditions to increase their interaction with the immobilized enzymes during their incubation period in the microreactor.
to the use of dialysis tubing: at 0 "C in five changes, 1L each, of the pH 7 phosphate buffer, and 45 min between changes. Biotinylation and m y t Purification of Trypsin. A p proximately 0.6 mg of benzamidine, followed by 2.7 mg of trypsin, was dissolved in 0.660 mL of triethanolamine buffer (0.1 M, pH 8.5, 0.01 M CaC12, and 0.1 M in NaCl) at 0 "C. Similarly, 0.7 mg of sulfeNHSLC-biotinwas dissolved in 0.24 mL of the same buffer EXPERIMENTAL SECTION Chemicals and Reagents. Water was distilled and deionized at 0 "C and rapidly mixed with the trypsin solution. The reaction (Millipore, Bedford, MA). Ammonium acetate, citric acid, glacial mixture was maintained at 0 "C for a period of 2 h. Dialysis of acetic acid, formic acid, ammonium hydroxide, sodium bicarbonthe reaction mixture was performed by the microtechnique of ate, sodium periodate, calcium chloride, triethylamine (TEA), Overall,13 at 0 "C in 1 L volumes with 45 min between changes. ethylenediaminetetraaceticacid (EDTA), dimethyl sulfoxide, ethTwo changes of Tris buffer (0.1 M, pH 7.2,O.Ol M CaC12, and 0.1 ylene glycol, acetone (Fisher Scientific, Fairlawn, NJ), sodium M NaCl) were followed by two changes of sodium acetate buffer acetate, Tris, triethanolaminehydrochloride,betaine, benzamidine, (0.1 M, pH 4.5, with 0.1 M NaCl). The final dialysis step used immobilized p-aminobenzamidine (Sepharose), hexadimethrine two changes of Tris buffer (0.1 M, pH 7.2, 0.01 M CaC12, and 0.1 bromide (Polybrene), sodium azide, a1-acid glycoprotein,oxidized M NaCl) to equilibrate with the binding buffer prepared for afsnity insulin B-chain (Sigma, St Louis, MO), (3aminopropyl)triethoxchromatography. Approximately 1 mL of immobilized pamiysilane (APTES), triethylamine (Aldrich, Milwaukee, WI) , nobenzamidine/Sepharose was placed into a 5 mL plastic syringe NeutrAvidin, sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS body (Becton Dickinson & CO., Rutherford, NJ) with a glass fiber LC-biotin), biotin-E-hydrazide (Pierce, Rockford, IL), &[[&[(lie frit to retain the affinity matrix. The resulting column was tinoyl)amino]hexanoyl]amino]hexanoic acid succinimidyl ester equilibrated with binding buffer (0.1 M Tris, pH 7.2,O.Ol M CaCl2, (NHSXX-biotin; Molecular Probes, Eugene, OR), trypsin (TPCK and 0.1 M NaCl) at 4 "C before applying the biotinylated trypsin. treated; Worthiigton Enzymes, Freehold, NJ), pepsin, and carAfter the biotinylated trypsin solution entered the affinity matrix, boxypeptidase-Y (CPY; Boehringer Mannheim, Indianapolis, IN) 12 mL of the binding buffer was passed through the matrix to were used as received unless otherwise indicated. wash unbound material. Bound trypsin was eluted with 6 mL of Supplies and Equipment. The coupled capillary CZE instru0.1 M acetic acid containing 0.02 M CaC12. This volume was ment was described in an earlier report." Fused-silica capillary placed in a centrifuge filter unit (Micron Separations Inc.) and (50 pm i.d., 360 pm o.d., Polymicro Technologies, Phoenix, AZ) concentrated on a centrifuge to approximately 0.3 mL. The was used in the preparation of the microreactors and for CZE affinity-purified biotinylated trypsin was then dialyzed overnight separations. An electric engraving tool was purchased locally in 1 L of sodium acetate buffer (0.1 M, pH 4.5 with 0.01 M CaC12) @remel, Racine, WI). at 0 "C. Biotinylationof Pepsin. Approximately 5 mg of pepsin was Preparation of Enzyme-Modified Microreactor. The prodissolved in 0.375 mL of sodium acetate buffer (0.1 M, pH 5.5, cedure described previously for the immobilization of trypsin at and 0.15 M NaCl) in an ice bath at 0 "C. The oxidation of pepsin the capillary inner surface8 has been m o d ~ e d . Fused-silica oligosaccharide with sodium metaperiodate was performed accapillaries were rinsed with various solutions by dipping one end cording to the method of O'Shannessy and Wilchek.12 Dialysis into the solution and then attaching the other end to the house of the reaction mixture took place in four changes of the sodium vacuum. After derivatization of the silica surface with APTES, acetate buffer, 1Leach, over a period of 24 h at 0 "C. Afterward, distilled, deionized water was used to rinse a 50 cm length of the oxidized pepsin solution was rapidly mixed with 0.125 mL of capillary for a period of 6 h to remove physically adsorbed material. the sodium acetate buffer containing 0.9 mg of biotin-LC-hyThe capillary was then purged by a nitrogen flush before rinsing drazide. The reaction proceeded at 25 "C for a period of 2 h after with several capillary volumes of dimethyl sulfoxide containing which the reaction mixture was dialyzed at 0 "C in five changes 0.5%TEA (v/v). NHSXX-biotin was dissolved in the 0.5%TEA of sodium acetate buffer (0.05 M, pH 4.5), 1 L each, with 45 min solution to a concentration of 5 mg mL-l, and several capillary between changes. There were losses and dilution of the pepsin volumes were drawn through the capillary at 30 min intervals over solution due to handling during transfers to and from dialysis a period of 6 h. The capillary was purged by a nitrogen flush membrane tubing in this procedure. The final biotinylated pepsin before rinsing with an ammonium acetate buffer (50 mM, pH 5, concentration was estimated as 1-5 mg mL-l. sodium azide 0.02%)for a 30 min period. Avidin was dissolved in Biotinylationof CPY. Approximately 2 mg of CPY dissolved the same acetate buffer to a concentration of 1mg mL-', and after in 0.3 mL of buffer was dialyzed overnight at 0 "C, in 1L of sodium application of vacuum to initiate flow in the capillary, the flow was phosphate buffer (0.05 M, pH 7, and 1 mM EDTA) before sustained for a period of 12 h at 4 "C by raising one end of the exchanging with 1 volume of pH 7.6 phosphate buffer (1 mM capillary by 15 cm. After the capillary was reattached to the EDTA and 0.02% sodium azide) for 1 h of dialysis. A 0.2 mg vacuum line, the capillary was rinsed with acetate buffer for a 30 sample of sulfeNHSLC-biotin was dissolved in 0.24 mL of the min period to remove unbound avidin. Before introducing the pH 7.6 phosphate buffer at 25 "C and rapidly mixed with the CPY biotinylated enzyme solution into the capillary, the capillary was solution. The reaction proceeded at 25 "C for a period of 3 h rinsed with a buffer solution to ensure stability of the particular before 0.2 mg of glycine was added to quench the remaining enzyme to be immobilized. Sodium acetate buffer (0.1 M, pH biotinylation reagent. Dialysis of the reaction mixture was 4.5) was used for pepsin, sodium acetate buffer (0.1 M, pH 4.5, performed by the microtechnique of Overall,13as an alternative 0.01M CaC12, and 0.02%sodium azide) was used for trypsin, and ammonium acetate buffer (0.05 M, pH 7, with 1 mM EDTA, and (12) O'Shannessy, D. J.; Wilchek, M. Anal. Biochem. 1990,191, 1-8. 0.02% sodium azide) was used for carboxypeptidase-Y. The (13) Overall, C. M. Anal. Biochem. 1987,165,208-214. Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
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biotinylated enzyme solution was then introduced into the capillary exactly as had been done with the avidin solution. Finally, the capillary microreactor was rinsed with the same buffer for 30 min to remove unbound material before storing the complete microreactor at 4 "C. Pepsin Microreactor: Digestion of Oxidized Insulin BChain. Initially, the pepsin microreactor was rinsed with several volumes of a 5%formic acid solution at 25 "C. After rinsing, one end of the microreactor was placed in 0.2 mL of a freshly made insulin B-chain solution, 0.5 mg mL-l in 5%formic acid at 25 "C. By raising this sampling end of the microreactor 15 cm above the free end, hydrodynamic flow was used to elute several microreactor volumes. The pepsin microreactor was immediately coupled to the separation capillary, and digestion of insulin proceeded for a period of 3 h at 25 "C before on-line analysis of the microreactor contents. The procedure to couple an enzymem o d ~ e dmicroreactor with a separation capillary has been described previously." Pepsin Digestion of Oxidized Insulin B-Chain in Homogeneous Solution. A 0.15 mg sample of insulin was dissolved in 0.05 mL of 5%formic acid solution and mixed with 0.025 mL of the biotinylated pepsin solution in a 50 mM sodium acetate buffer, pH 4.5. This homogenous mixture was allowed to react in a temperaturecontrolled bath at 35 "C for a period of 24 h before analysis. CPY Microreador Digestionof Oxidized Insulin B-Chain. The CPY microreactor was rinsed with several volumes of an ammonium acetate digestion buffer (0.05 M, pH 6.5,l mM EDTA, and 0.02%sodium azide) at 25 "C. A 0.2 mg sample of oxidized bovine insulin B-chain was dissolved in 0.2 mL of the acetate buffer, and one end of the microreactor was placed into the B-chain solution. Again, hydrodynamic flow (15 cm height differential) was used to rinse several column volumes of the insulin solution through the microreactor. Flow through the microreactor was halted by placing both microreactor ends into a sterile rubber septum. The microreactor and contents were placed into a Petri dish attached to an electric engravingtool which served as a means of inducing vibration in the microreactor for approximately an 11 h period (overnight). After 22 h, the CPY microreactor was removed from the septum and coupled with a Polybrene-modified separation capillary for on-line analysis of the microreactor contents. A similar procedure was simultaneously carried out with an avidin-mod~edcapillary (no CPY present) that served as the control for nonspecific fragmentation. In turn, the avidin-only modified capillary was placed into the coupled system for on-line analysis of its contents. Trypsin Microreactor Digestion of Denatured al-Acid Glycoprotein. The al-acid glycoprotein (AAG), 2 mg mL-l, was denatured in Tris buffer (0.1 M, pH 8.1) for 24 h at 100 "C.I5The trypsin microreactor was manipulated exactly as described for the CPY-modified microreactor to accomplish a digestion of the denatured glycoprotein. The glycoprotein residence time in the microreactor was 12 h at 25 "C. A control for nonspecific fragmentation of the glycoprotein was simultaneously prepared under identical conditions with an avidin-modified capillary (no trypsin present) and the glycoprotein solution. After approximately 11 h of residence time, the trypsin microreactor was (14) Nashabeh, W.; El Rassi, 2. J. Chromatogz 1992,596, 251-264. (15) Schmid, IC In AlpharAdd Glycoprotein: Genetics, Bwchemist*y, F%~i~logical Functions, and Phurmacolow, Braumann,P.,Ed.; Alan R Liss, Inc.: New York, 1989 p 11.
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Analytical Chemistty, Vol. 66,No.24, December 15, 1994
coupled to a Polybrenemodifiedseparation capillary and the digest was analyzed exactly as described for the CPY microreactor. In turn, the avidin-modified capillary was placed into the coupled system for on-line analysis of its contents. Capillary Zone ElectrophoreticSeparations. CZE separations of the pepsin hydrosylate of oxidized insulin B-chain and B-chain solutions were performed in APTESderivatized fusedsilica capillaries (a descriptionof the AITES derivahtion appears above, i.e., preparation of the microreactor). Before using the AF'TES capillaries for separations,the columns were equilibrated with the electrophoresisbuffer (5%formic acid) for approximately 30 min to remove any physisorbed APTES. The homogeneous digests were sampled with hydrodynamic injection. On-line sample injection of the pepsin microreactor hydrosylate was accomplished after the capillary junction was filled with a 0.5% formic acid solution and the separation capillary had been filled with a 5% formic acid solution. The pepsin microreactor and separation capillary were aligned in the well with a solution gap of 100 pm. A potential of -5 kV was applied for 45 s across both the microreactor and separation capillary to electrokinetically transfer an aliquot of the hydrosylate to the CZE capillary. Immediately afterward, the 0.5%formic acid was replaced with the electrophoresis buffer (5%formic acid) to induce a separation after application of -240 V cm-' across only the separation capillary segment. CZE analyses of the trypsin hydrosylate of denatured AAG were performed in fused-silica capillaries that had been washed with 0.1 M NaOH and distilled water before dynamic modification with a cationic polymer. A 2%Polybrene solution with 2%added ethylene glycol was rinsed through the capillary for 30 min, followed by deionized water for 10 min and equilibration with the electrophoresis buffer for 10 min. The homogenous digest was introduced by hydrodynamic flow (15 cm height differential for 1 min). On-line sample injection of the trypsin microreactor hydrosylate (or the AAG control from an avidin-modified microre actor) was accomplished after the capillary junction well was filled with a 10 mM ammonium citrate buffer (PH 3.2) and the separation capillary had been filled with the electrophoresis buffer (PH 3.2, 50 mM citric acid with 250 mM betaine). The trypsin microreactor and the separation capillary were placed into the well and aligned with a solution gap of either 50 or 100 pm. The sample was transferred by applying a potential of -15 kV across both microreactor and separation capillaries for either 75,45, or 30 s. Immediately after sample introduction, the 10 mM buffer in the well was replaced with the electrophoresis buffer to enable the separation upon application of -370 V cm-' to the separation capillary. CZE separations of the CPY hydrosylate of oxidized insulin Bchain were performed in fused-silica capillaries that had been washed with 0.1 M NaOH and distilled water before dynamic modification with Polybrene (as above). Homogenous digests were introduced by hydrodynamicflow (15 cm height differential for 1 min). On-line sample injection of the CPY hydrosylate (or the B-chain control from an avidin microreactor) occurred after the capillary junction well was filled with a 5 mM ammonium formate (PH3.8) buffer and the separation capillary had been filled with a 50 mM ammonium formate buffer (PH 3.8). The microre actor and the separation capillary were placed into the well and aligned with a solution gap of 75 pm. A potential of -5 kV was applied across both microreactor and separation capillaries for
75 s to transfer an aliquot of the hydrosylate (or the Bchain solution from the control microreactor) to the CZE capillary. Immediately afterward, the buffer in the well was replaced with the 50 mM separation buffer to initiate the separation upon application of -320 V cm-l to the separation capillary.
7
INJECT
H
V POWER SUPPLY
RUN
r*
1
I*
ENZYWE-MODIFIED
SEPARATION
RESULTS AND DISCUSSION
The selectivity of CE separations of nanoliter samples can be greatly enhanced by adding an enzyme catalyst to picomole or smaller quantities of analytes prior to their separation and d e t e c t i ~ n . ~ The . ~ J ~specificity of the enzyme offers an additional dimension for analyte identification (e.g., for the identification of single variants in the primary sequence of a protein or for unmasking an analyte of interest on the electropherogram). Immobilization of this catalyst allows its application to be more reproducible in repeated analyses and prevents contamination of the sample with the enzyme or its autolytic products. In this work, we describe a variety of enzyme-mediatedchemistries interfaced with on-line CZE analyses, including digestion of proteins, removal of amino acids from the carboxy termini of the protein, and peptide mapping of a heavily glycosylated protein. With only minor changes, the avidin-biotin system described earlier for the immobilization of trypsin onto a fused-silica capillary has been utilized to immobilize pepsin and CPY for use in on-line protein analyses. The immobilization procedure using avidin-biotin chemistry is assembled at the capillary inner wall in three steps. Briefly, treatment of the fused-silica capillary wall with APTES provides free amino groups on the surface of the 50 pm i.d. capillary. Biotin was covalently attached to these free amines through the formation of an amide bond. Avidin is then bound to the surface-immobilized biotin, and the binding between biotin and avidin is so strong (& = M) that it can tolerate extreme conditions of temperature, pH, and different solvent systems. M e r incubation with avidin, the remaining biotin-bindingsites on each surface-tethered biotin-avidin complex are available to bind a biotin-conjugated enzyme. This design allows easy variation of the enzyme used for the microreactor, and the exceptionally mild conditions during its attachment to the capillary wall assures maximal retention of activity. Immobilization of a variety of biotinylated proteases in this design has permitted a number of heterogeneous proteolytic degradations to be utilized for protein characterization at the subpicomole level of sample. This will make these traditional means for structural analyses of proteins more amenable to the combined requirements of extremely limited availability of samples and increasing protein structural complexity. Fast peptide mapping in a coupled trypsin microreactor/ capillary electrophoresis separation system was demonstrated previously.*1A fluid junction allows for replacement of the solution in the gap between the coupled capillaries while the two capillary segments remain unmoved (Figure 1). The fluid associated with the transfer of solutes between the two segments differs from the CZE separation buffer primarily in ionic strength, generally being 1order of magnitude more dilute than the separation buffer. Online sample introduction is facilitated by matching electroosmotic flow in the two segments through either covalent or dynamic modification of the capillary wall of the separation capillary, but (16) Grant, D.L.;Martin, W. G.; Anastassiadis, P. A.J. Bid. Chem. 1967,242, 3912-3918. (17) Chien, R-L.; Helmer, J. C. Anal. Ckem. 1991,63,1354-1361.
TEFLON CELL COUbLlNo UNIT
1-
CZE SEPARATION CAPILLARY
ENZYME WODlFlED CAPILLARY
Figure 1. Schematic diagram of the coupled capillary CZE instrument. Two segments of 50 pm i.d. fused-silica capillary (one 50 cm segment used as a microreactor, the other 75 cm segment used as the separation column for CZE) are coupled via a fluid joint. Inset: Detailed schematic of the Teflon cell in which the digestion and the separation capillaries were coupled. One capillary was secured to the cell, while a three-axis Burleigh inchworm controller (roughly 50 nm step resolution) was used to align the end of the other capillary to within a predetermineddistance (50-1 00 pm) of the first capillary. A stereomicroscope was used to visually inspect the positioning of the two capillaries. Other details are given in the text.
the lengths of the capillary segments may differ. This allows online sample introduction by electromigration of an aliquot of protein hydrosylate across the fluid gap onto the CZE capillary. This was accomplished on-line in the coupled system for protein hydrosylates from several different enzymemodified capillary microreactors. The buffer composition and pH was varied independentlyin the two capillary segments of the coupled system to optimize the enzyme activity and separation efficiency of the CZE analyses of the enzyme-catalyzed reaction products. The two schemes used for the immobilization of the proteolytic enzymes are shown in Figure 2. Either primary amines present on the exterior of trypsin and CPY (scheme 1) or oligosaccharides present on CPY and pepsin (scheme 2) are modilied by covalent attachment of biotin through a long chain spacer arm. Prior to their immobilization in the microreactors, the activity of these biotinylated enzymes had been qualitatively examined by comparison of peptide maps obtained with the biotinylated and nonbiotinylated enzymes. M e r immobilization of the enzymes in the microreactors, the catalyzed reactions were apparent from selective degradations of proteins relative to a control medium in which the enzyme was absent. The activity of the pepsin-modified microreactor was also evaluated with respect to the biotinylated enzyme in free solution. A comparison of peptide maps generated by the microreactor reaction and a homogeneous solution reaction using biotinylated pepsin is shown in Figure 3. The top electropherogram was obtained after hydrodynamic injection of Bchain hydrosylate from the homogeneous solution reaction mixture. The lower electropherogram was obtained in a coupled pepsin microreactor-CZE system after on-line injection of an aliquot of Bchain insulin digest. Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
4403
Scheme 1 )
Enzyme - NH,
NHS LC B i o t i n
>
0 II
Enqme-Nn-C-
LC Biotin
Scheme 2) H,O l?H Enzyme- CHCH,->En2yme--CHO
Blotin-LC Hydrazide
> Enzyme-HHN= CH-LC Eiotln
Figure 2. Schematic representation of the immobilization of proteolytic enzymes to the inner surface of a fused-silica capillary. Either primary amines present on the exterior of trypsin and CPY (scheme 1) or an oligiosaccaride present on CPY and pepsin (scheme 2) are modified by covalent attachment of biotin through a long chain spacer arm followed by attachment of avidin and then the respective biotinylated enzyme. See text for details.
The nearly identical profiles of the peptide maps shown in Figure 3 would indicate the immobilized pepsin had similar activity relative to the dissolved biotinylated pepsin. Another conclusion drawn from these peptide maps is that the immobilized pepsin and the solubilized biotinylated pepsin show similar preferences for peptide bond cleavage sites within the B-chain peptide. The large surface area to volume ratio (800 cm-l) within a 50 pm i.d. enzyme-modifed fused-silicacapillary microreactor minimizes the diffusional limitations on the reaction rate and provides a high effective enzyme concentration within the microreactor.' The activity of enzymemodified microreactors has been estimated on the basis of comparison of the heterogeneous (microreactor) hydrolysis reaction with that of the homogeneous reaction. Qualitatively, we have examined both the number of peptide fragments generated in each case and the efficiency of digestion (Le., the relative proportion of the intact protein before and after reaction). The high catalytic efficiency of the enzyme microreactor results in a rapid degradation of amorphous proteins (e.g., b-casein and large linear peptides) under gravity-flow conditions in the microreactor. A 50 cm length, pepsin-modified microreactor produced the fragmentation, shown in a off-line CZE analysis of the Bchain peptide of bovine insulin (Figure 4, top). The Bchain insulin sample had a transit time through the microreactor of approximately 30 min. Limited fragmentation of globular protein samples (e.g., cytochrome c, myoglobin, and hemoglobin) and glycoprotein samples (e.g.,AAG) has indicated that extending the incubation period as long as 24 h did not allow sufticient interaction of these complex protein structures with trypsin. Relevant reaction parameters for enzyme-mediated hydrolysis include (1) time of reaction, (2) temperature of reaction, (3) enzyme concentration and catalytic efficiency, and (4) mass transport of sample. Each of these parameters was adjusted to obtain greater fragmentation of these more dacult protein structures. In particular, we have attempted to increase temperature, transport of solutes, and loading of active enzyme into the 4404
Analytical Chemistry, Vol. 66,No. 24, December 15, 1994
E
0
2
4
6
8
10 12 T i m (mln)
14
16
18
20
Figure 3. Comparison of homogeneous and immobilized pepsin on the hydrolysis of 6-insulin. (Top) Electropherogram of the homogeneous pepsin hydrolysate of oxidized insulin 6-chain, 2 mg mL-I in 100 mM NH4HC02, pH 2.0, 20:l (w/w) ratio, 35 "C, 24 h reaction time. Hydrodynamic injection 10 s,at 10 cm relative height. Separation voltage, -15 kV in 5% H2C02, capillary, 46 cm to detector. (Bottom) Electropherogramobserved for an on-line analysis of oxidized insulin 6-chain, 0.5 mg mL-I in 5% H2C02, following 3 h residence time in a pepsin-modifiedmicroreactor. Before each run, -25 kV was applied for 45 s over the coupled system to transfer samples across a 100 pm solution gap into an ATPES-treated capillary (50 pm i.d., 360 pm o.d., 54 cm length). The fluid junction was composed of 0.5% H2C02 during injection. Separation conditions were identical to above.
capillary microreactor. Temperatures as high as 40 "C did not appear to promote the degradation of globular protein samples using the trypsin microreactor (data not shown). This may be an indication that kinetic limitations to the rate of hydrolysis had not become dominant Puritication of the biotinylated trypsin with affinity chromatography was performed immediately before proceeding with the microreactor immobilization in these experiments to increase the catalytic efficiency of the enzyme. In meeting the criterion for possessing specific binding ability, the ptdied enzyme fraction had increased the likelihood that catalytically active trypsin would occupy the surface immobilization sites. The overall strategy for improving catalytic efficiency in the trypsin microreactor attempted to combine higher loading of the active enzyme with an increased interaction of substrates with the enzyme. Most importantly, the transport rates for the delivery of substrate to the enzymemodified capillary wall may be
I
0
2
10 12 14 16 16 20 (dn) Figure 4. Comparison of off-line pepsin microreactor digestion of oxidized b-insulin with control. (Top) Pepsin hydrosylate of 2 mg mL-’ 8-chain of bovine insulin, collected after 30 min period of gravity flow through pepsin-modified microreactor; 15 s hydrodynamic injection onto APTES-derivatized capillary with sampling end at 25 cm height relative to detection end of capillary; 5% formic acid solution as electrophoresis buffer and -260 V cm-I (18PA current) applied in separation; absorbance read at 214 nm. (Bottom) A 10 s hydrodynamic injection of the 2 mg mL-’ 8-chain insulin solution (native protein) onto APTES-derivatized capillary with sampling end at 25 cm height relative to detection end of capillary;5% formic acid solution as electrophoresis buffer and -360 V cm-I (24 yA current) applied in separation; absorbance read at 214 nm. 4
6
8
increased by adding convective transport to the diffusional transport of the solutes. This can be accomplished in the microreactor during incubation of proteins by simply placing the microreactor in contact with a vibrating surface during the incubation period. Addition of these steps to the digestion procedure improved the overall success rate dramatically, especially concerning the larger, more complicated proteins. Better than 50%of the microreactors used in this manner successfully digested proteins as complex as AAG. Improvements in the catalytic efficiency of the trypsin-moaed microreactor have made it possible to integrate the enzymemediated hydrolysis of complex protein structures with on-line CZE analysis. A demonstration of the on-line procedure for a CZE analysis of tryptic fragments of bovine al-acid glycoprotein is shown in Figure 5. This model glycoprotein is of particular interest because of its very high carbohydrate content, consisting of oligosaccharides, either linear or branched, that account for approximately 40% of the molecular weight (approximately 40 OOO) .I6 The AAG is denatured prior to its introduction into a trypsin microreactor (and an avidin-only microreactor control), which were externally in contact with a vibrating surface during an 11h incubation period. Significant hydrolysis of the denatured AAG was observed when a trypsin-modified microreactor was employed
(Figure 5). The absence of fragmentation in the avidin-only control microreactor was observed after four injections from the control (data not shown), where a small peak for the transferred AAG was evident, but no additional peaks were observed. The on-line sampling of the digest of AAG from the trypsin microreactor was evaluated in a series of consecutiveCE analyses, each preceded by an on-line injection from the microreactor (Figure 5). These injections were for 75 and 45 s and twice for 30 s periods. The fluid in the junction was replaced before and after each injection; however, the capillary segments remained undisturbed but for minor alignment of the capillary mouths before each injection. After the second injection for 30 s, removal of the microreactor from the junction verified the absence of any effect on the ensuing separation due to its close proximity to the separation capillary during the previous run. After inspection of the peak profiles in the four CE analyses of AAG hydrosylate, two features of the peptide maps that accompany the decreases in injection times are identitiable. There are decreases in the areas of the peaks and the resolution improves, most significantly, for earlier eluting peptides. The decreasing peak areas are consistent with electromigration of smaller sample volumes onto the separation capillary during shorter injection times. The resolution improvement may be attributed to the combined influences of decreases in the length of the sample zone injected onto the capillary and decreasing dilution by the low conductivity gap buffer inadvertently injected with the sample. The conditions used for electrophoretic fluid transfer of the trypsin microreactor sample allow for sample stacking10$18 to occur due to the concentration and pH changes as the sample migrates from digestion buffer to gap buffer to separation buffer. The largest quantities of the digest were observed after the 75 s injection period without evidence of dilution of the sample or peak broadening due to stacking. The l@folddilution of the gap buffer with respect to the digestion and CZE separation buffers promotes sample stacking across the dilute gap buffersg In an optimal injection of the microreactor sample, a minimal plug of the dilute gap buffer enters the separation capillary and there is minimal change in the 5 potential of the capillary wall and its Polybrene modification in the vicinity of the injection. The conductivity differences between zones containing the transferred sample, the gap buffer, and the CZE separation buffer can result in local electroosmotic flows that differ from the bulk electroosmotic flow in the separation ~api1lary.l~ This situation can produce hydrostatic pressures across the conductivity boundaries positioned within the CZE capillary during and after the injection period, which lead to significant losses in res01ution.l~Dilution by the gap buffer during on-line injections for periods of 1min or longer can be minimized by careful attention to experimental parameters. These include precise alignment of the capillary ends, the conductivity differences between the gap buffer and the electrophoresis buffer, the length of the injection time, the solution gap width, the magnitude of the applied voltage during the injection, and differences in the electroosmotic flow in the two capillary segments. We have also investigated the utility of integrating CPYmediated hydrolysis of the carboxyl terminal of proteins with onl i e CZE analysis of the hydrosylate. Analysis of the C-terminal sequence of proteins using the exopeptidase CPY has received much interest since its introduction because of the lack of reliable (18) Aebersold, R; Momson, H.D.J. Chromatogr. 1990, 516,79-88.
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Figure 5. Effect of duration of on-line injection on separation efficiency of the tryptic digest of AAG. Four electropherograms show successive on-line injections and separations in the coupled system of a tryptic digest of aj-acid glycoprotein (2 mg mL-j) in pH 8.1, 100 mM Tris-HCI. Before each run, -1 5 kV was applied over the coupled system to inject samples into the Polybrene-modifiedseparation capillary (50pm i.d., 360 pm o.d., 62 cm length). During each run, the applied field was -370 V cm-1 and the electrophoresis buffer was 50 mM citric acid-250 mM betaine, pH 3.2: (a) 75 s injection; 20 mM ammonium citrate pH 3.2 in the fluid junction; 50 pm solution gap in coupled system. (b) 45 s injection; 20 mM ammonium citrate pH 3.2 in the fluid junction; 100 pm solution gap in coupled system. (c) 30 s injection; 10 mM ammonium citrate pH 3.2 in the fluid junction; 100 pm solution gap in coupled system. (d) 30 s injection with a 100 pm solution gap of 15 mM ammonium citrate pH 3.2 in fluid junction. After this injection, the microreactor was removed from the fluid junction before the separation of the transferred digest was initiated.
non-enzymatic methods to sequentially remove amino acids from the C-terminus.2° CPY was chosen over other exopeptidases for sequencing the C-terminus of peptides and proteins because of its ability to free most amino acid residues from the C-termini of proteins.21 This allows the production of a ladder of truncated peptides, which can be used for sequencing or identification of the parent p r ~ t e i n . The ~ ~ *major ~ ~ problem evident in previous work utilizing CPY for the production of these ladders utilizing (19) Chien, R-L.; Burgi, D. S. Anal. Chem. 1992,64,489A-496.k (20) Hayashi, R; Moore, S., Stein, W. H.J.Bid. Chem. 1973,248,2296-2302. (21) Hayashi, R Methods Enzymol. 1976,45, 568-587.
4406 Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
either homogeneous or heterogeneous digestion conditions involves the variation in the relative hydrolysis rates for the removal of different amino acids can produce digests of varying composition over time." Considerable variation in the homogeneous digestion of the Bchain of bovine insulin was observed by CZE after incubation with biotinylated CPY for variable intervals (22) Tsugita, A; Van den Broek, R; Przybylski, M. FEBS Left 1982,137,1924. (23) F'ilosof, D.;Kim, H. Y.; Dyckes, D F.;Vestal, M. L. Biomed. Mass Specfrom. 1984,11,403-407. (24) Hayashi, R; Bai, Y.; Hata, T./. Biochem. 1975, 77,69-79.
two electropherograms shown represent consecutive injections of the digest from the CPY-modified microreactor demonstrating the reproducibility of the injection from the same digest. Electropherograms for the injection of Bchain insulin sampled from the avidin-only modhed microreactor (control) did not show evidence of fragmentation (Figure 6, bottom). The use of immobilized CPY in a capillary microreactor will allow the CPY digest to be sampled at various intervals to allow a thorough investigation of the reaction matrix as a function of time. This will allow a more reliable characterization of the CPY digestion products, since intermediate forms of the truncated peptides may appear and disappear depending on the reaction kinetics of the subsequent step in the hydrolysis of very small amounts of protein. CONCLUSIONS
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Figure 6. On-line CPY digest of insulin B-chain versus avidin-only control. (Top) Two successive electropherograms showing an online injection and separation of a CPY digest of oxidized insulin B-chain (1 mg mL-l) in 50 mM, pH 6.5 ammonium acetate buffer. Before each run, -5 kV was applied for 75 s over the coupled system to inject samples across a 75 pm solution gap into the Polybrenemodified capillary (50pm i.d., 360pm o.d., 62 cm length). The buffer in the fluid junction was composed of 5 mM, pH 3.8 ammonium formate during the on-line injection. During each separation, the field applied was -320 V cm-l and the electrophoresis buffer was 50 mM ammonium formate, pH 3.2. (Bottom) Two successive electropherograms collected in a coupled microreactor system using an avidinonly modified microreactor (control; no CPY present) which had received the same insulin B-chain solution. The characteristics of the fluid junction, sampling time, applied voltage across coupled system, and separation conditions were identical in each case.
of from 1 to 24 h (data not shown). CPY was immobilized in a capillary microreactor and this was utilized in an on-line system to produce digests of the insulin Bchain. The electropherograms in Figure 6 represent the on-line analysis of the CPY-digested insulin B-chain after a 24 h reaction period. Large quantities of the truncated peptides appear using a 75 s injection, indicating the extensive hydrolysis of B-chain insulin in the CPY-modified microreactor (Figure 6, top). The
The proteolytic enzymes we have utilized in the integrated microreactor-separation capillary design are distinguished by the large differences in their specificities toward peptide bond cleavages and their pH of greatest catalytic efficiency. The analytical procedure for degradation of structurally complex proteins in the trypsin-modified microreactor and transfer of an aliquot by electromigration to the separation capillary has been optimiied to obtain the tryptic peptide maps of a heavily glycosylated protein. The quantity of the tryptic fragments injected into the separation capillary and the resolving power of the ensuing electrophoretic separation have each shown a dependence on the length of the injection period. Optimal transfer of truncated peptides from a carboxypeptidase-Y modzed microreactor to the CZE capillary demonstrates the capability for regulating dilution of the injected sample by the gap buffer. Carboxypeptidase-Y and pepsin each possess high catalytic activity in the presence of high concentrations of denaturants (urea and hydrogen ion, respectively), and the high catalytic efficiencies of these in the analytical procedure demonstrated here holds much promise for analysis of complex protein structures. ACKNOWLEDGMENT
This work was supported, in part, by a National Science Foundation Presidential Young Investigator Award (Grant CHE897394), and by a Grant provided by Procter and Gamble Corp. The donations of the Inchworm Controller by Burleigh Instruments, the UV detector by Applied Biosystems, Inc., and NeutrAvidin from Pierce Chemical Co. are gratefully acknowledged. Received for review August 8, 1994. Accepted October 13, 1994.@ @
Abstract published in Advance ACS Abstracts, November 1, 1994.
Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
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