Anal. Chem. 1992, 64, 1610-1613
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Trypsin-Modif ied Fused-Silica Capillary Microreactor for Peptide Mapping by Capillary Zone Electrophoresis Lawrence N. Amankwa and Werner G. Kuhr’ Department of Chemistry, University of California, Riverside, California 92521
An analytical procedure Involving tryprln Immoblllzed on the Inner surface of a 50-jm-1.d. fused-silica capillary has been developed for on-line digestion of minute amounts of protein. Trypsin was hnmoMllzed onto the surface of an amlnoalkylsilane-treated fused-slllca capillary via biotin-avldln-blotln coupling. The enzyme-modtfledcapillary was used to dbest /%casein simply by flowing a solution of the denaturedprotein through the capillary at a rate of 40 nUmln and collectlng the effluent. A 50lengthof capillary was long enough to obtain complete dlgestlon of &casein In about 25 mln at room tomperature. The activity of the enzyme on the capillary was high enough to ensure complete and continuow digestion of protein over a period of 12 h. Separation of the tryptk pep tldes In the cdlected digest was accomplkhed by capillary zone electrophoreds, CZE. Highly efficient reproducible separation of all the tryptic peptides was obtalned wlng only 14 fmol of InJecIed digest (from a total dlgest of about 100 pmd). Laser-lnduced fluorescence was used to detect the separated NDA-labeled tryptic peptides In the digest.
INTRODUCTION Enzymatic hydrolysis of a protein followed by separation and detection of the individual peptide fragments in the hydrolysate remains a powerful analytical technique for the identification and characterization of proteins.1s2 Traditionally, enzymatic hydrolysis reactions are performed in homogeneous solutions consisting of a mixture of the proteolytic enzyme and the protein under investigation. It is desirable to maintain optimum conditions of pH, temperature, protein: enzyme ratio, and reaction time to achieve efficient and reproducible hydrolysis. However, the use of enzymes in homogeneous solutions has several disadvantages. Enzymes often lose their catalytic activity fairly rapidly leading to lack of reproducibility in the fragmentationpattern. Additionally, in order to maintain an optimum enzyme:protein ratio efficient hydrolysis can only be performed on large (greater than microgram) quantities of substrate. To overcome disadvantages associated with homogeneous enzymes, Cobb and Novotny3s4have utilized trypsin immobilized on agarose gel for the characterization of phosphorylated and dephosphorylated @-casein.Immobilized enzymes are considerably more stable and retain their catalytic activities for much longer times than the free enzymes. It has also been reported that enzymes immobilized into a carbohydratepolymer retain their catalytic activity even in some organic solvents.5 The ability to easily separate immobilized enzymes from the reaction mixture offers an additional advantage of multiple use of the (1) Ingran, V. M. Nature (London) 1956,178, 792-794. (2) Moon, K.; Smith, E. L. J. Biol. Chem. 1973,248 (9), 3082-3088. (3) Cobb, K. A.; Novotny, M. Anal. Chem. 1989,61, 2226-2231. (4) Guilbault, G. G. Analytical Uses of Immobilized Enzymes; Marcel Dekker Inc.; New York, 1984; Chapter 2. ( 5 ) Wang, P.; Hill,T. G.; Wartchow, C. A.; Huston, M. E.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D., Callstrom, M. R. J.Am. Chem. SOC. 19922,114, 378-380. 0003-2700/92/0364-1610$03.00/0
enzyme resulting in reduced cost. Immobilized enzyme reactors are also compatible with the analysis of very minute quantities (nanogramamounts) as well as very small volumes (nanoliters to microliters) of protein solutions. The development of immobilized enzyme reactors is therefore essential for microscale identification and characterization of proteins. A wide variety of analytical separation techniques have been used for the separation of peptide fragments in the hydrolysis reaction. These techniques range from slab gel electrophoresis,Bthin-layer and reversed-phase high-performance liquid ~ h r o m a t o g r a p h y ? and ~ ~ ~capillary ~*~ gel and capillary zone electrophoresis?l”l2 With the decreasing size of protein samples that are available for characterization there is an increasing need for very sensitive analytical separation techniquesfor the analysis of proteins. Among the separation techniques mentioned above only microcolumn HPLC and CZE have the requisite efficiency and sensitivity for analyzing picomole amounts of protein hydrolysate. Separation of peptide fragments by CZE is less complex and less time consuming than by microcolumn HPLC which often requires the use of solvent gradients. CZE is therefore rapidly gaining ground as the separation technique of choice for analysis in microscale protein research. The focus of our research has been to develop an analytical technique involving an integrated enzyme microreactor for fast peptide mapping of very minute amounts (femtomole to picomole) of protein by CZE with minimum sample preparation and handling. The enzyme microreactor consists of a 50-cm-long fused-silica capillary (50-pm i.d.1 onto which trypsin is immobilized on the inner surface. It was possible to obtain a complete tryptic digest of &casein using this reactor simply by flowing the protein solution through the capillary. This report discussesthe preparation of the enzyme microreactorby coupling the enzyme to the capillary wall via biotin-avidin-biotin coupling. In addition, the characteristica of the reactor and application of the reactor to the digestion of a test protein and subsequent separation of the peptide fragments in the digest by CZE will be discussed.
EXPERIMENTAL SECTION Immobilizationof Trypsin onto a Fused-Silica Capillary. An appropriate length of 50-pm-i.d., 360-pm-o.d fused-silica capillary tubing (PolymicroTechnologiesInc., Phoenix, AZ)waa cleaned by perfusing (by vacuum suction) with 0.1 M NaOH for 10 min followedby a 10-min rinse with distilled water. Residual water waa removed by purging with air for 20 min. The cleaned capillary waa then perfused with a 2% acetone solution of (3aminopropy1)triethoxysilanefor 30 min followed by a 10-minair (6) Lu, H.S., Lai, P.H. J. Chromatogr. 1986,368,216-231. (7) Fuller, C. S.; Wasaerman, R. H. J. Biol. Chem. 1979, 254, 72087212. (8)Stephen, R. E. Anal. Biochem. 1978,84, 116-126. (9) Cohen, A. S.; Karger, B. L. J. Chromatogr. 1987,397,40+417. (10) Jorgenson, J. J.; Lukaa, K.D.J. Chromatogr. 1981,218,209-216. (11) Groesman, P. D.; Colburn, J. C.; Lauer, H.H.; Neilsen, R. G.; Riggin, R. M.; Sittampalan, G. S.; Rickard, E. C. Anal. Chem. 1989,61, 1168-1194. (12) Albin, M.; Wiktorowicz, J. E.; Black, B.; Moring, S. Am. Lab. 1991, Oct, 27-31. 0 1992 American Chemlcel Soclety
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
purge and incubation at 45 "C overnight. Following this treatment, the capillary was perfused with a 5.0 mg/mL solution of NHS-LC-biotin in 0.05 M N&C03 (pH 8.6) for 4 h at room temperature. After this treatment the capillary was rinsed with water and then treated again by flowing (gravity) a 4 mg/mL solution of avidin in 0.05 M sodium phosphate buffer (pH 7.4) through it at a rate of about 40 nL/min for 24 hat 4 "C. Finally, the avidin-coated capillary was rinsed with distilled water and then treated (by gravity flow) for 24 h at 4 O C with a 10 mg/mL solution of biotin-labeled trypsin in sodium phosphate buffer. Unbound trypsin was rinsed (by vacuum suction) from the capillary with about 10 column volumes of buffer (50 mM sodium phosphate, pH 7.4) and stored in the refrigerator until ready to use. Preparation of Tryptic Digest. A 3 mg/mL solution of 8casein in 0.050 M ammonium bicarbonate buffer (pH 8.0) was boiled for 10 min in a water bath to denature the protein. A 150-pL aliquot of the resulting solution was placed in a 200-pL glass sample vial made locally from a Pasteur pipet. An appropriate length of the enzyme-coated capillary was cut and rinsed with ammonium bicarbonate buffer and one end of the capillary was placed in the sample solution. The other end was connected to a vacuum line to fill the capillary with sample solution. The capillary was carefully disconnected from the vacuum while solutionwas still slowlyflowing through it by gravity siphoning. Under these conditions, the flow rate of the protein solution through the capillary was measured to be about 40 nL/ min. The effluent (Le., digest) from the capillary was collected in another sample vial until an adequate volume (- 20 pL) was acquired. Digestion using trypsin immobilized on agarose gel was done simply by running &casein solution through a Pasteur pipet which contained 1mL of trypsin-modified gel, where the gel was trapped using a silanized glass wool filter at the tapered end. Digests were either analyzed immediately or stored in the refrigerator at 4 OC until ready for analysis. CZE Analysis of Digest. The CZE apparatus used in this work was built locallyand has already been describedin an earlier report.ls Simply, it consists of a high-voltage power supply (150 kV; Glassman High Voltage Inc., Model PSEH50R02.0, Whitehouse, NJ) connected to the anodic buffer reservoir, which is isolated in a Plexiglas enclosure with an interlock to ensure operator safety. A fused-silicacapillary (PolymicroTechnologies, Phoenix, AZ)100cm in length (50-pm i.d., 360-pm 0.d.) was used as a separation capillary. The capillary was cleaned with 1.0 M NaOH followed by a distilled water rinse prior to use. Injection was accomplished by electromigration at 25 kV for 1.0 s, and timing was controlled by an electronic counter. All separations were performed at an applied voltage of 25 kV unless otherwise stated. Detection was achieved on column by focusing the 442nm (50.6 mW) output of a HeCd laser (OmnichromeModel 20568/25M,Chino, CA) after passing through a quartz Brewster prism (No. ABSU-15, Optics for Research) and a 1-cm focal length quartz lens, onto a spot (25 cm away from the low-voltage end of the separation capillary) that had been cleared (by heat) of the polyimide coating. The fluorescence generated was imaged through a microscope objective and a spatial and two long pass glass color filters (Schott, No. G6495) onto a photomultiplier tube (Hamamatsu, R928). Data were acquired on a strip chart recorder (Model EV41-885835, Kipp & Zonen, Holland). All samples were fluorescently labeled with NDA" prior to analysis. Fluorescent labeling was performed by mixing a 20-pL volume of the digest with 50 pL of 0.1 M NaCN, 100pL of 0.01 M sodium phosphate, pH = 9.6 and 10p L of a 2 mg/mL methanol solution of NDA and allowing the mixture to stand at room temperature for at least 10 min. m e n t e . Water was distilled and deionized (Millipore,Bedford, MA). Sodium bicarbonate,ammonium bicarbonate,sodium phosphate (monobasicand dibasic),acetone, and methanol were obtained from Fisher Scientific(Fairlawn,NJ). &Casein,trypsin (biotin-labeled),FITC-ExtrAvidin,and ExtrAvidin were obtained from Sigma (St. Louis, MO). NHS-LC-biotin (sulfosuccinimidyl 6-(biotinamido)hexanoate)and immobilized TPCK-trypsin (13)Amankwa,L.;Scholl,J.;Kuhr, W. G.Anal. Chem. 1990,62,21892193. (14)Roach, M. C.;Harmony, M. D. Anal. Chem. 1987,59,411-415.
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Flgure 1. A schematic diagram showing the blndlng vla bbtln-avldln of an enzyme onto the surface of a fused-slllca caplllary.
on agarose gel were obtained from Pierce (Rockford, IL). Sodium cyanide (Mallinkrodt, Paris, KY), (3-aminopropy1)triethoxysilane (3-APTS)(AldrichChemicalCo., Inc., Milwaukee, WI), and 2,3-naphthalenedialdehyde(NDA) (Molecular Probes, Inc., Eugene, OR) were used as received without further purification. Tris-phosphate buffer (75 mM, 10% methanol, pH = 7.6) was donated by Applied Biosystems, Inc., San Jose, CA.
RESULTS AND DISCUSSION Immobilized enzyme reactors have extensively been used for a variety of biochemical analysis and chemical diagnosis.4J5J6 This is a direct consequence of the fact that immobilization of enzymes offers several advantages over solution enzymes. Generally, immobilization of an enzyme makes it more resistant to unfolding of its native structure caused by effects of heat and pH since the environment of the enzyme is similar to that in which it exists naturally." Immobilized enzymes are also less susceptible to effects of activators and inhibitors and thus more useful as analytical reagents. There are various methods of immobilizing en~ymes.~J5J* However a highly specific and extremely strong noncovalent binding of an enzyme to a solid support is by the biotinavidin couple.lgZ0 The strength of the nonspecific binding M) that it between biotin and avidin is so strong (& = can tolerate extreme conditions of temperature and pH and different solvent systems. In this work, biotinylated trypsin (DPCC treated) waa immobilized onto the inner surface of a fused-silica capillary which has previously been treated successively with 3-APTS, biotin, and avidin. The 3-APTS treatment provides free amino groups on the surface of the capillary to which biotin waa covalently attached through an amide bond. Treatment of this biotin-bound capillary with avidin fiially yields a surface to which the biotinylated trypsin can be attached, as shown in Figure 1. The efficiency of the interaction between the surface-bound biotin and avidin was investigated with fluorescence microscopy. To accomplish this, the surface modification reaction (treatment with 3-APTS, biotin, and avidin) was performed on the outside of a fused-silica capillary which had been stripped of the polyimide coating and the aminated surface was dipped in a solution of FITC-labeled avidin. For comparison a second capillary that had not been treated with 3-APTS and biotin was similarly treated with FITC-labeled avidin. The fluorescence emission from each of these capillarieswas measured with a fluorescencemicroscope (Zeiee Axioskop) equipped with a CCD camera (Thompson 7895 B containing a 521 by 512 CCD chip) and a Photometrics NU200 controller. Data were acquired and analyzed with a Mac IIci computer. The samples were excited with a 488-nm line of a 200-W Hg arc lamp. Shown in Figure 2 are the 3-D emission spectra obtained from the surfaces of the capillaries. The intensity of fluorescence is indicated by the height of (15)Weetall, H. H.Anal. Chem. 1974,46,602A-615A. (16)Ngo, T.T.Int. J . Biochem. 1980,11,459-465. (17)Martinek, K.;Klibanov, A. M.; Goldmacher, V. S.; Berezin, I. V. Biochem. Biophys. Acta 1977,485,1-12. (18)Shufang, L.; Walt, D. R. Anal. Chem. 1989,61,1069-1072. (19)Uditha De Alwis; Wilson, G. S. Talanta 1989,36(1,2),249-253. (20)Gunaratna, P.C.;Wilson, G. S. Anal. Chem. 1990,62,402-407.
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Flgure 3. Electropherogram showing the separation of NDA-labeled @-caseinin a 50 mM Tris-phosphate buffer, pH = 7.9. Conditions: separation capillary 50-pm Ld., 360-pm o.d., 100 cm (75 cm to detector) long: applied potential, 25 kV; current, 10.0 pA. Sample (14.0 pM) was injected for 10 s at 25 kV. Fluorescence detection using the 442” line of a He-Cd laser. The peak at the elution time 12.5 min is due to impurities in the derhratization reagents.
B
Figure 2. 3-D fluorescence image of the outer surface of FITCExtrAvMin-modiiedfused-silicacapillaries obtained with a fluorescence microscope interfaced with a CCD camera. Fluorescence intensity is indicated by the height of the peaks in the image. (Bottom) representative image of FITCIxtrAvidin bound to a fused-silicacapillary which had earlier been treated with aminoalkylsilane(APTS)and (NHSLC-Biotin). (Top) image of a control fused silica that had been treated with only FITC-ExtrAvidin.
peaks in the third dimension. The distribution of the fluorescence intensity indicates the distribution of avidin on the surface. Figure 2A is that for the nonbiotinylated capillary, while, Figure 2B is for the biotinylated capillary. Fluorescence emission from the nonbiotinylated capillary accounts for only emission from nonspecifically adsorbed FITC-labeled avidin. The lack of surface features in Figure 2A indicates that very little avidin was nonspecifically adsorbed on the surface of the capillary. The rich surface structure illustrated in Figure 2B suggests the presence of a large amount of avidin, thus confirming the specific interaction between biotinlabeled surface and FITC-avidin. The immobilization technique as depicted in Figure 1 localizes the trypsin further away from the capillary surface, making it possible for it to interact freely with substrate. The exact amount of trypsin immobilized on the surface is not known;however, by assumption of 1:l binding of trypsin to avidin (Note: each molecule of avidin has four biotin binding sites) and with the cross-sectional area of avidin which is about 2000 A2,2l the surface of a 50-pm-i.d., 50-cm-long capillary can hold only 6.5 pmol of trypsin at saturation. Direct
immobilization of trypsin onto the surface of the capillary offers advantages such as low-pressure drop across the capillary which makes it applicable to CZE electroosmotic generated flow conditions. The relatively large surface area to volume ratio (800 cm-1) within the capillary enables adequate interaction of the flowing substrate with the surfaceimmobilized trypsin. Application of the Trypsin-Modified Capillary to Protein Digestion. @-Caseinwas used as a test protein mainly because its tryptic map has been well documented.3 Trypsin has a unique property as a proteolytic enzyme since it hydrolyzes specificallya t only the C-terminalsides of lysine and arginine residues. Tryptic digests are therefore less complex. A 50-cm length of the trypsin coated capillary was used to obtain a tryptic digest for @-casein. The flow rate of the protein solution through the capillary was maintained at about 40 nL/min to allow enough time for the protein to interact with the enzyme. At this flow rate the interaction time was calculated to be a t least 25 min since the totalvolume of the capillary is about 1 pL. Figure 3 shows the electropherogram of NDA-derivatized @-casein. The peak at the elution time of about 12.5 min is due to impurities in the reagents. The broad shape of the protein peak (elution time = 27 min) is due to the fact that a large amount (1.2 pmol) of the protein was injected. In order to check for evidence of digestion, electropherograms of the protein were obtained prior to and after perfusion through the trypsin-modified capillary. Figure 4 is the electropherogram showing the separation of a &casein digest. Perfusion with protein solution was performed continuously for 16 h, during which time a totalamount of about 5 nmol of @-caseinwas completely digested by the trypsin-immobilized capillary. The amount of digest actually injected into the separation capillary was about 14 fmol. Most of the 16 tryptic fragmentsin the digest can clearly be resolved; however no attempt was made to identify them. To confirm that the peaks in Figure 4 were indeed due to the tryptic peptides, a similar digestion of @casein was made using TPCK-trypsin immobilized on agarose gel. The CZE separation of this digest is shown in Figure 5. The similarity between the peak profiles in Figures 4 and 5 suggests that our trypsin-coated capillary has more than satisfactory activity. The efficiency of the trypsin-coated microreactor for protein digestion was tested by varying the length of the capillary used. Under the present conditions even a 10-cm length of (21) Weber, P. C.; Ohlendrof, D. H.; Wendoloski, J. J.; Salemme, F.
R. Science
1989,243,8588.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
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I
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Flgure 4. Electropherogram showing the separation of NDA-labeled tryptic digest of &casein (dlgestlon performedwith trypsin lmmoblllred onto the surface of a bO-cmlong, 50-pm-1.d fused capillary). InJection was performed for 1.O s at 25 kV. Other separation conditlons were as given In Flgure 3.
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Figure 6. Electropherogramshowing the separation of NDA-labeled tryptic digest of &casein (dlgestlon performedwith trypsln lmmoblllzed onto the surfaceof a 10-cmlong, 50-pm1.d. fused capillary). InJection was performed for 1.O s at 25 kV. Other separation conditlons were as given in Figure 3.
fl
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Flgurr 5. Electropherogramshowing the separation of NDA-labeled tryptlc digest of @-casein (dlgestlon performedwlth trypsln Immobllized on agarose gel). Injectin was performed for 1.0 s at 25 kV. Other separatlon conditions were as given in Flgure 3.
trypsin-modifiedcapillary has adequate activity to digest picomole quantities of substrate. Figure 6 shows the CZE separation of a digest obtained using only a 10-cm-long trypsin-coated capillary. All the peaks correspondingto the tryptic fragmente can be identified. A closer examination of the baseline (between elution time 20 and 30 min), however, shows a slight hump which is due to undigested &casein. Because the interaction time between the immobilized trypsin and the substrate in this case was only about 5 min in the 10-cm capillary, total digestion can only be achieved for very small amounts (less than 25 pmol). The use a 50-cm length of capillary, however, results in complete digestion of all of the protein perfused through it. This is a significant improvement in enzyme catalysis time over earlier applications of open tubular heterogeneousenzyme reactors22 which required very long lengths in order to achieve complete enzyme catalysis. This improvement is attributed to the relatively large surface area:volume ratio of our enzyme reactor. Also, the trypsin-coated capillary maintained its activity for weeks when stored (with the ends sealed to prevent drying) in the refrigerator at 4 O C . In fact, a properly stored capillary can be used for at least three consecutive 12-h digestion periods without significant loss of activity. The immobilized approach used in this work resulted in two significant changes in the interfacial properties of the fused-silica capillary. First, the treatment of the capillary with (3-aminopropy1)triethoxysilanereplaces the surface silanol groupswith amino groups. The presence of the inherent negative surface charge on the original fused-silica capillary is therefore convertedto a net positive charge. Consequently, (22) Inmam, D.J.; Hornby, W.E.Biochem. J. 1972,129, 255-262.
there is a net reversal in electroosmoticflow when a potential is applied at the ends of the capillary. Thus, it is necessary to reverse the polarity of the applied potential to elute solutes under electrophoretic conditions. The second effect of the immobilization of trypsin onto the capillary is that solutes (proteins, peptides, and amino acids) severely adsorb onto the capillary resulting in very poor solute recovery and long elution times as well as very severely distorted elution peaks. Attempts to eliminate solute adsorption by capping the adsorption sites by running 0.5% aqueous solution of glutaraldehyde for 30 min followed by a 10 mM glycine did not result in an improvementin protein recovery from the enzyme reactor.
CONCLUSION The work presented in this report demonstrates that a hydrolytic enzyme such as trypsin can be readily immobilized onto the surface of a fused-silica capillaryto produce an open tubular heterogeneous enzyme reactor for on-column digestion of very small amounts of protein for subsequent characterization by peptide mapping. Immobilization of the enzyme via the biotin-avidin-biotin couple produces a highly stable and catalytically active capillary that can be used for complete digestion of picomole quantities of protein. The poor recovery of solutes from the capillary however, limits its application to the digestion of proteins in amounts greater than 10-12 mol. An interesting aspect of this work is that the enzymemodified capillary can easily be coupled directly to the separation capillary to enable on-line protein digestion and separation with very little sample handling. Data on this aspect of this work will be presented in a future report.
ACKNOWLEDGMENT This work was supported, in part, by the National Science Foundation (Grant No. CHE-897394)and by Grants provided by Eli Lilly Research Laboratory and the Procter and Gamble Co. Donation of the HeCd laser by Omnichrome Corp. is gratefully acknowledged. RECEIVED for review February 10, 1992. Accepted April 20, 1992. Registry No. Trypsin, 9002-07-7; (3-aminopropy1)triethoxysilane, 919-30-2;NHS-LC-biotin,109940-19-4;2,3-naphthalenedialdehyde, 7149-49-7; silica, 7631-86-9.
