Ami. Chem. lW3, 65, 2093-2697
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TECHNICAL NOTES
On-Line Peptide Mapping by Capillary Zone Electrophoresis Lawrence N. Amankwat and Werner G. Kuhr' Department of Chemistry, University of California, Riverside, California 92521
INTRODUCTION Recently, we described an analytical procedure for immobilizing a proteolytic enzyme (trypsin) on the inner surface of a 50-pm4.d. fused-silica capillary for on-line digestion of picomole quantities of protein.' Trypsin was immobilized onto the surface of an aminoalkylsilane-treated fused-silica capillary via biotin-avidin-biotin technology. Subsequently the enzyme-modified capillary was used to digest &casein simply by flowing a protein solution through it a t a rate of 40 nWmin and collecting the effluent. This effluentcontained the peptide fragments of the protein, which could be separated by capillary zone electrophoresis (CZE) to yield the tryptic map for characterization and identification of the protein. This method has numerous advantages over traditional homogeneous methods used for digesting proteins:2 (1)the enzyme-modified fused-silica microreactor can be used for repeated digestions; (2) the eluted digest is totally free from contamination by the proteolytic enzyme; (3) the small size of the reactor makes it especially compatible for use in the analysis of small quantities (picomole) of protein as well as for analysis of small volumes of samples (typically, a few nanoliters). These advantages are particularly important when one is ascertainingsubtle differencesin protein structure (e.g., differences of a single residue between homologous proteins or differences resulting from posttranslational modifications). 3 9 4 In our previous application of this enzyme-modified capillary reactor, the effluent (digest) from the reactor was collected in a microvial before reinjection onto a separation column for subsequent separation of the peptide fragments by CZE. It is obvious that this step, the collection of the effluent, will not only be impractical but will also be highly susceptible to sample loss and/or contamination, especially when one is confronted with the analysis of nanoliter-scale samples. Thus, it would be of great practical utility to be able to perform the digestion on-line with the separation of the digest. Several investigators have also explored the possibility of performing enzyme-mediated chemistry on-line with an electrophoretic separation. Nashabeh and El &si6 have described a technique involving enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary electrophoresis. Guzman et al. have used an on-line concentrator-reactor cell to perform digestions of nanogram quantities of protein on-line with a CZE separation.6 The f Current address: University of British Columbia, Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia, V6T 123 Canada. (1) Amankwa, L. N.; Kuhr, W. G.Anal. Chem. 1992,64,16lC+1613. (2) Cobb, K.A.; Novotny, M. Anal. Chem. 1989,61,2226-2231. (3) Hubennan, A.; Aguilar, M. B. J. Chromatogr. 1988,443,337-342. (4) Wheat, T. E.; Young, P. M.; Astephen, N. E. J. Liq.Chromatogr. 1991,14,987-996. (5) Nashabeh, W:; El h i , Z. J. Chromatogr. 1992,596, 251-264. (6) Guunan,N. Sixth Annual SymposiumoftheProteinSociety1992, T-92. Guzman, N.; Hernandez., L.; Advis, J. P. HPCE'93 1993, W-58.
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coupling configuration described in these systems does not allow the use of different buffer components and/or pH's during the enzymaticreaction stage and during the separation stage. This requirement is particularly important in many enzyme reactions, since the pH required for optimum catalytic activity of the immobilizedenzyme is generally different from that required for the separation of products from the reaction. This is especially true for digestions involving trypsin, since it has the greatest catalytic activity a t p H s between 8 and 9 while the best separation for the peptide digest lies in the lower pH range (pH 2-4h4 The simplest way to use different buffers in the digestion and separation processes is to break the system into two distinct parts and couple the two capillaries together via a fluid joint. Numerous techniques for coupling micron-size fused-silicacapillarieshave been reported in the literature?-11 Unfortunately, none of these designs could be used for such an on-line sample introduction since it is important to have both quantitative mass transfer of sample across the joint when the sample is injected and complete isolation between the sample and the separation capillaries during the electrophoretic Separation. Recently, we described a simple technique for transferring a solute across a wide capillary junction (ca. 50-200 pm).12 The two capillaries were placed in close proximity by careful micropositioning, and the distance between them was controlled to submicron resolution. Very little diffusional sample loss in the gap was observed a t low ionic strength, since the voltage gradient across the gap is large and the sample had little time for radial diffusion out of the junction. The utility of this approach is that it not only offers efficient mass transfer of the sample by electromigration but should also allow subsequent separation of the transferred sample in the second capillary without the need to move either capillary. In this report, we have used the technique described above for coupling our enzyme-modified capillary reactor to a CZE fused-silica capillary for use for on-line protein digestion and separation of the digest by CZE. The enzyme-modifiedfusedsilica microreactor was coupled through a 100-pm solution gap to the separation capillary. First, the enzyme-modified capillary was filled with the protein solution and allowed to incubate at room temperature for -2 h, during which time proteolysis occurs. Subsequently, an aliquot of the digest was injected onto the separation capillary by applying a potential across the two free ends of both capillaries. The injected sample was then separated by applying the CZE (7) Olefuowicz, T. M.; Ewing, A. G.Anal. Chem. 1990,60,1872-1876. (8) Wdingford, R.A.; Ewing,A. G. Anal. Chem. 1987,69,1762-1766. (9) Pentoney, S. L.; Huang, X.; Burgi, D. S.; Zare, R. S. A d . Chem. 1988,60,26262630. (10) Nickerson, B.; Jorgenson, J. W. J. Chromatogr. 1989,480,157168. (11) Albin, M.; Weinberger, R.; Saap,E.; Moring, S. Anal. Chem. 1991, 63,417-422. (12) Kuhr,W. G.;Licklider, L.; Amankwa, L. Anal. Chem. 1993,65, 275-282. Q 1993 Amerlcan Chemical Soclety
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previously been treated successively with 3-APTS. biotin, and avidin. The 3-APTS treatment provided free amines on the surfaceofthecapiUary,allowingthecovalentattachmentofbiotin through an amide bond. This was accomplished by perfusing the capillary with a 5.0 mg/mL solution of NHS-LC-biotin in 0.05 M NaHC03(pH 8.6) for 4h a t room temperature. After this treatment the capillary was rinsed with water and then treated with a 4 mg/mL solution of avidin in 0.05 M sodium phosphate buffer (pH 7.4) at a rate of -40 nL/min for 24 h at 4 "C using gravityflow. Finally, theavidin-coatedcapillary wasrinsed with distilledwaterandthentreatedfor24hat4oCwitha10mg/mL solution of biotin-labeled trypsin in sodium phosphate buffer. Unbound trypsin was rinsed (by vacuum suction) from the capillary with -10 column volumes of buffer (0.05 M sodium phosphate, pH 7.4)and stored in the refrigerator until ready to use. This procedure produces a layer of trypsin bound to the surface of the capillary without exposing trypsin to the harsh chemicals necessary for the silica modification, thereby greatly increasing the viahility of the surface-bound enzyme. Finally, a 10 mM aqueous solution of glycine was flowed through the digestion capillary for 10min in order to block adsorption sites introduced during the enzyme immobilization process in the preparation of the reactor. This treatment has significantly reduced the effectsof adsorption of small molecules, as reflected in shorter elution times of amino acids and peptides. However, no significant change in the migration of proteins was observed through the column. This might be expected, since proteins bind with some affinity to the active site of trypsin, which is on the Flgare 1. Schematicdiagramoftlh?coupledcapllialy CZE Instrument. capillary surface. Twosegmentsof50-pmi.d.fusedslllca capilialy(one50cmsegment Coupling Microreactor and SeparationCapillaries. The usedasamicroreacta.meomer7Xmsegnentusedasthesepara~n coupling of the microreactor and the separation capillaries was column for CZE) were coupled vla a fluM joint. which wntalned ether accomplished in a specially designed and electrically insulated distilled water or a l0-lold dllution of th8 separation buffer. Inset: A Teflon cell (Figure 1, inset; total volume of -10 mL of buffer). detailed schematic of the Teflon cell in which the digestion and the One end of the enzyme-modified capillary reactor was rigidly separationcapillark were coupled. One capillary was secured to the connected to the Teflon cell. The separation capillary was call. while a three-axis Burleigh inchworm controller (roughly 50-nm mounted onto a three-axis micropositioner utilizing Burleigh step resolution)was used to align the end of the other capillary within inchworm motors (Burleigh, Fisher Park, NY). One end of the ether 1 (touchlng) or 100 pm of the end of the first capillary. A separation capillary was then inserted through a hole in this cell stereomicroscopewas used to visually inspect the positioning. Other such that one end was directly aligned with that of the enzymedetails are given in the text. modified capillary. The junction between the two capillaries was viewedthrough a stereomicroscopewith a help of a microscope separation potential across the gap solution and the other illuminator focused onto a diffusely reflective target located end of the separation capillary. Using this approach, we were underneath the Teflon cell. The separation between the two able to perform on-line digestion and separation of picomole capillaries was controlled withsubmicron resolution. The entire quantities protein by CZE in less than 3 h. Teflon cell housing the capillary junction was mounted on four Kel-F supports attached to a Plexiglas platform to ensure EXPERIMENTAL SECTION complete electricalisolationof the capillaryjunction fromground. Preparation Of Tryptic Digest. A 2 mg/mL solution of Chemicalsand Reagents. Water wasdistilled and deionized @-caseinin 0.050 M ammonium bicarbonate buffer (pH 8.0) was (Millipre, Bedford, MA). Sodium bicarbonate, ammonium boiled for 10 min in a water bath to denature the protein. A bicarbonate,sodium phosphate (monobasicand dibasic),acetone, 150-pL aliquot of the resulting solution was placed in a 200-pL ethylene glycol, and methanol were obtained from Fisher glass vial made from the tip of a Pasteur pipet. A 50-cm length Scientific(Fairlawn,NJ). Hexadimethrine bromide (Polybrene), of the enzyme-modified capillary was rinsed with ammonium @-casein,trypsin (biotin-labeled), and ExtrAvidin were obtained bicarbonate buffer, and one end was connected to the vial from Sigma (St.Louis, MO); sulfosuccinimidylB-(biotinamido)containing the protein sample. The free end of the microreactor hexanoate (NHS-LC-biotin)was obtained froinpierce (Rockford, was connected to a house vacuum line in order to fill it by suction IL);sodium cyanide (Mallinkrodt, Paris, KY), 3-(aminopropyl)with the protein solution. Subsequently, the vial containing the triethoxysilane,(BAITS;Aldrich ChemicalCo., Milwaukee,WI), protein solution was elevated relative to the other free end ofthe and 2,3-naphthalenedialdehyde(NDA; Molecular Probes, Inc., capillary reactor to ensure slow hydrodynamic flow (-40 nLI Eugene, OR)were used as received without further purification. min) of the protein solution through the reactor into another Supplies and Equipment. The CZE apparatus (Figure 1) vial. After -5 column volumes (5 pL) of the protein solution used in this work was built locally and has been described in an had flowed through the reactor, the free end of the capillary earlier report.13 Basically, it consists of a high-voltage power reactor was carefully connected to the Teflon cell while sample supply ( i 5 0 kV; Glassman High Voltage Inc., Model solution was still flowing through it, as shown in the inset of PSEH50R02.0, Whitehouse, NJ). The high-voltage end was Figure 1. Following this, the two ends of the capillary reactor isolated in a Plexiglas enclosure with an interlock on the door to were leveled and maintained at the same height to minimize any ensure operator safety. Fused-silica capillary (50-pm i.d., 360nonelectroosmotic flow. p m 0.d.; Polymicro Technologies, Phoenix, AZ) was used for Capillary ElectrophoresisSeparation.AllCZEseperationa construction of the enzyme-modified microreactor or as the were performed in fused-silica capillaries which were cleaned separation capillary. with 1M NaOH and distilled water prior to use. AU capillaries Trypsin-Modified Microreactor. A detailed description of were dynamically modified with a cationic polymer by flowing the procedurefor immobilizingtrypsinontoafused-silica capillary either a Microcoat solution (Aoolied Biosvstems. Inc.) or a 2% was given previously.' Briefly, biotinylated trypsin was immosolution of Polgbrene in 2 % etcileneglycoisolution through the bilized onto the inner surface of a fused-silica capillary that had capillary for 30 min, followed by a 5.min distilled water rinse and (13)Amank~a,L.;S~hall,J.;Kuhr.W.G.Anol.Chem.1990,62,218~ a IO-min equilibration with the appropriate separation buffer (9U mM sodium phosphate. pH 7 . 4 ~ . 2193. POWER SUPPLY
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 19, OCTOBER 1, 1993
Digest samples that were prepared off-line were injected into the separation capillary via electromigration. On-line injection of digests were accomplished as follows (Figure 1). Prior to online sample injection, the capillary was filled with the protein solution. Next, the ends of the capillary reactor and the separationcapillary were brought to within 100pm of each other. Injection was performed by applying a high voltage (-20 kV) across both capillaries for a preset time (-1 min). During this time, an aliquot of the digest in the capillary reactor was transferred across the solution gap between the two capillaries into the separation capillary. During injection,the buffer in the Teflon cell was a 10-folddilution of the buffer used for separation (yielding9 mM sodium phosphate, pH 7.4). After injection, the buffer surroundingthe capillary junction was carefully replaced with the separation buffer. The separation was then accomplished by applying a potential across only the separation capillary. Detection was either by fluorescenceor by UV absorbance. In cases where fluorescence was used, the peptides resulting from the tryptic digest were labeled with NDA prior to injection onto the separationcapillary. Fluorescence detectionwas performed as described previously. Briefly, the 442-nm (50.6-mW)output of a HeCd laser (Omnichrome,Model 2056-8/25M, Chino, CA) was focused with a 1-cm focal-length quartz lens onto a spot 10 cm from the downstream end of the separation capillary (which has been previously cleared of the polyimide coating). The fluorescence signal was imaged through a 1OX microscope objective, a spatial, and two long-pass glass filters (Schott, No. G6495) onto a photomultiplier tube (Hamamatsu, R928). UV absorbance detection was accomplished on a locally assembled UV detector, essentially consistingof components of the detector for an Applied Biosystems Model 270A UV detector. Data were recorded on a strip chart recorder (Kipp & Zonen).
RESULTS AND DISCUSSION This work deals with the on-line digestion, injection, and separation of peptide digests. By performing all these steps on-line, one will be able to overcome most of the typical problems such as sample loss and contamination that normally plague the analysis of minute quantities of protein samples. On-line digestion is particularly advantageous in peptide mapping because of the high reproducibility requirement of the maps, and also because of the ever decreasing sizes of protein samples that are becoming available to the medical, bioanalytical, and the biotechnologicalcommunities. Several other investigators have already explored the possibility of performing enzyme-mediated chemistry on-line with an electrophoretic separation. Guzman et al. have reported the use of an on-line concentrator-reactor cell to perform digestions of nanogram quantities of protein on-line with a CZE separation.6 Nashabeh and El Rassi6 have described a technique involving enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary electrophoresis. Ribonuclease T1, hexokinase, and adenosine deaminase were successfully immobilized on the inner walls of a short fusedsilica capillary through glutaraldehyde attachment. In that report, the enzyme-modified capillary reactor was coupled in series with a CZE separation capillary via a PTFE tube, where substrates were introduced as thin plugs into the enzymemodified capillary reactor. The catalyzed reaction occurred as the substrates migrated down the reactor either by hydrodynamic flow or by eledromigration pass the connection point. The joint was then disconnected, and the separation of the enzymatic reaction products was subsequently performed by immersing the separation capillary in buffer. It is important to note that the coupling configuration used in these reports does not allow the use of different buffer Componentsand/or pH’s during the enzymatic reaction stage and during the separation stage. This requirement is particularly important in protein digestion, since the pH required for optimum catalytic activity of the immobilized
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enzyme is generally different from that required for the separation of products from the reaction. This is especially true for digestions involvingtrypsin, since it has the greatest catalytic activity at pH’s between 8 and 9 while the best separation for the peptide digest course in the lower pH range (pH 2-4).4 The simplest way to use different buffers in the digestion and separation processes is to break the system into two distinct parts, and couple the two capillaries together via a fluid joint. Numerous techniques for coupling micron-size fused-silica capillaries have been reported in the literature. Most of these designs involve mass transfer across a “tight electrically conductive joint” which was first developed for use with postcolumn electrochemical detectors for CZE.6-8 Although, mass transfer across these joints (from one capillary segment to another) has been shown to be highly quantitative, mass transfer of buffer from the bathing solution into the capillary has proven to be very difficult because of the short distance between these joints (typically
