Anal. Chem. 1002, 64,879-888 (3) KGnig, W. A. The Recti98 of Enantlomw Separetbn by Capllary Oes Chrometogrephy;Hvthi: Heidelbecg, 1987. (4) Schurlg, V. J . Ckomtogr. 1988,441, 135. (5) Frank, H.; Nicholson, G. J.; Bayer, E. J . Chromatogr. Scl. 1977, 75, 174. (6) Smdkova-Keulemansova, E. J . Chrometogr. 1982,257, 17. (7) Koscielskl, T.; Sybllska, D.; Jurczak, J. J . Chrometogr. 1983,280, 131. (8) Khig, W. A.; Lutz, S.; Wenz, G.; von der Bey, E. H@h Res. Chromatogr. Chfomogr. Commun. 1988, 7 7 . 508. (9) SchuriQ, V.; Nowotny. H. P. Angew. Chem., Int. Ed. Engl. 1990,29, 939. (10) Berthod, A.; Ll. W. Y.; Armstrong, D. W. carbahydr. Res. 1990,207, 175. (11) Hen. S. H.; Armstrong, D. W. In Chkel Separetions by H R C ; Krstulovic, A. M., Ed.: Ellis Hotwood Limlted: Chlchester. 1989; ChaDter 10, pp 208-284. (12) Armstrong, D. W. Anal. Chem. 1987,59, 84A. (13) Armstrong, D. W.; LI, W. Y.; Chang, C. D.; Pltha, J. Anal. Chem. 1990. ... 62. .- 914. . (14) Ll, W. Y.; Jln. H.-L.; Armstrong, D. W. J . Chromatogr. 1990, 509, 303. (15) Armstrong, D. W.; LI, W. Y.; Stalcup, A. M.; Secor, H. V.; Izac. R. R.; Seeman, J. I. Anal. Chlm. Acta 1990,234, 385. (16) Ll, W. Ph.D. Dkrsertatlon, Unhrerslty of Mlssouri-Rolla, 1990. (17) Kosclelskl, T.; Sybllska. D.; Felt, L.; Smolkova-Keulemansova,E. J. J . Chromatogr. 1984,286, 23.
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(18) Andera, L.; Smolkova-Keulemansova, E. J. J . Inclusion &nom. 1987,5,397. (19) Ettre, L. S. The Kovats Retention Index System. Anal. Chem. 1964, 36. - - , 31A. - .. .. (20) Watabe, K.; Charles, R.; OICAv, E. Angew. Chem., Int. Ed. €ngl. lgag. 28. 192. -(21) Schurig. V.; Osslg, J.; Link, R. Angew. Chem.. Int. Ed. Engl. 1989, 28, 194. (22) Koppenhoefer, B.; Bayer, E. Chromatographia 1984, 79, 123. (23) Lumty. R.; Rajender, S. Bispolvmes 1970.9 , 1125. (24) Leffbr, J.; Grunwald, E. R8t8S end Equlllbrle or Orgenlc RaecHons; Wiley: New York, 1963. (25) Melander. W.; Campbell, D. E.; Horvath, C. J . Chromatogr. 1978. 758, 215. (26) Krug, R. R.; Hunter, W. G.; m r , R. A. J . phvs. Chem. 1978,80, 2335. (27) Krug, R. R.; Hunter, W. G.; Grbger, R. A. J . Fhys. Chem. 1978,80, 2341. (28) Armstrong, D. W.; Nome. F.; Spino, L. A.; Golden, T. D. J . Am. Chem. Soc.1986, 708, 1410. (29) Smolkova-Keulemansova, E. J.; SoJak, L. ACS Symposium Ser. 1987, 342.247.
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RECEIVED for review September 12,1991. Accepted January 22, 1992.
Peptide Mapping of Complex Proteins at the Low-Picomole Level with Capillary Electrophoretic Separations Kelly A. Cobbt and Milos V. Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A varlety of dyfereni pepWe-mapplng schemes are plesented,
wlth emphark on the development of procedures whlch can be done wlth llmlted quantltks (Le. 5 pmol) of proteln. Resub are OMaIned hwn model proteins whkh contah dzsuMde bonds, whlch must be broken prlor to fragmentatlon of the proteln. A reactlon lnvolvlng the dmultaneous use of trC butylphosphlna and Smethylazlrldlne to reduce and alkylate the dkufflde bonds Is employed, due to favorable attrlbutes of these reagents for the rcaleddown procedure. The tradltlonal performlc acld oxldatlon reactlon to cleave cystlne groupr k ako wccessfully u w d wlth lowglcomole quantltles of proteln. Three dmerent proteh d@stbn reagents are u w k trypeln, chymotrypsin, and cyanogen bromkle. Each reagent produces a unlque mlxture of peptldes. Caplllary electre phorerk Is used to separate the peptMer, offerlng hlgh sep aratlon efflclencles, short analydr times, and compatlblllty wlth small sample slzer. I n addttlon to the conventional use of UV detectlon for underlvatlzed peptldes, laser-Induced fluorescence detectlon Is employed In conjunctlon wlth an arglnlne-solecllve derlvatlzatlon reactlon. Thls latter procedure for derlvatlzatlon and detectlon offers an alternatlve peptklamapplngmode, In whlch only the arglnlnacontalnlng peptides are detected, and b useful In sknplltylng the peptlde maps of large protelns.
INTRODUCTION The study of proteins frequently involves the use of peptide mapping, a powerful and efficient means of protein charac*To whom all corres ondence should be addressed. Present address: Tfe Dow Chemical Co., 1897 Building, Midland, MI 48667.
terization or identification. The basic premise of peptide mapping is to enzymatically or chemically cleave a protein into a number or smaller peptide fragmenta and then separate the resulting peptides, either chromatographically or electrophoretically, to yield a characteristic peak profile or “map” of that particular protein. Peptide mapping is primarily a qualitative, comparative technique, and ita popularity stems from the ability to ascertain very subtle differences between proteins, such as amino acid substitutions or posttranslational
modification^.'-^ Over the years, a number of sample treatment procedures have been developed for the purpose of peptide mapping. Most often, these procedures have been designed for micromole or nanomole quantities of protein.&l0 However, there is a growing need for the ability to obtain reproducible and informative peptide maps from much smaller amounts of protein, down to the picomole (or nanogram) level. This need arises from the fact that many proteins of interest to the medical or bioanalytical community, such as those occurring in physiological fluids, cell surface receptors, or growth fact o r ~ are , ~ typically ~ isolated in extremely s m d quantities. To be able to obtain needed information from such proteins, it is necessary that reliable procedures be developed for reduced-scale sample treatment. We have previously addressed some of the needs pertaining to high-sensitivity peptide mapping by demonstrating the use of immobilized trypsin for the digeation of as little as 50 ng of protein, followed by peptide separation using either capillary electrophoresis (CE)or microcolumn liquid chromatography.12 However, our previous studies were performed on proteins with relatively simple tertiary structures, with no disulfide bridges. Because many proteins of interest contain disulfide linkages, which tend to complicate the sample treatment procedure for peptide mapping, it is imperative that the methods be expanded to
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be able to effectively deal with all types of proteins. Peptide mapping of complex proteins involves several sample preparation steps necessary to obtain the desired peptide fragments in a reproducible manner. First, the protein must be denatured or suitably unfolded to expose any potential reaction or cleavage sites. Second, disulfide bonds, if present, should be eliminated, generally by reduction and alkylation13 or by oxidation14 of the cystine groups. Third, the protein is fragmented by a site-selective cleaving reagent such as trypsin, chymotrypsin, or cyanogen bromide.I5 The fourth and final stage is the separation and detection of the resulting peptides to yield the peptide map. In dealing with low-picomole quantities of protein, the challenge is primarily one of micromanipulation of the sample,l1J6or being able to carry out the necessary reactions and separations while sample loss is minimized and the required reproducibility is maintained. In this paper, we report a series of procedures which have been developed for the purpose of high-sensitivity peptide mapping in a variety of different modes. We have used model proteins which contain several disullide bridges to demonstrate the applicability of the procedures to large or more complex proteins. Cleavage of the disulfide bonds has been accomplished in two ways, either reduction and alkylation with trib~tylphosphine'~-~~ and 2-methyla~iridine:+~ respectively, or oxidation using performic acid.24 Three different means of fragmenting the proteins have been used: immobilized trypsin, immobilized chymotrypsin (both used in the reactor column format, as described in ref 12), and cyanogen bromide. Capillary electrophoresis (CE) is the separation method employed for all results shown here. CE has proven to be highly effective in the resolution of many different sample typea, including peptides,sB proteins,*s oligonucleotides,ws For carbohydrates,37-39and a variety of smaller molecules.our purposes in high-sensitivity peptide mapping, CE provides the efficient separations necessary for the complex peptide mixtures, and is compatible with very small sample volumes. Results are presented using both W absorbance detection, in which all peptides in the mixture are represented, and laser-induced fluorescencedetection, used in comjunction with the selective derivatization of only the arginine-containing peptide^.'"^' This latter variation of peptide mapping can be very useful when the map of a large protein is too complex to be easily interpreted with UV detection, which detects all peptides in the mixture and can result in an overwhelming number of peaks. By derivatizing only certain peptides in the digest mixture with a fluorescent tag, the resulting peptide map is less crowded, easier to evaluate, and yet is still very representative of the original protein. All of the peptide-mapping procedures presented here have been specifically developed to be suitable for use with very small quantities of protein, taken through all necessary sample preparation and separation steps. The reproducibility of the sample treatments and separation methods, an area of particular concern due to the comparative nature of peptide mapping, is addressed.
EXPERIMENTAL SECTION Equipment. The capillary electrophoresissystem used in this study was a modular unit, assembled in-house. The high-voltage power supply, capable of delivering 0-30 kV, was purchased from Spellman High Voltage Electronics Corp. (Plainview, NY). UVabsorbance detection was performed with a variable-wavelength UV detector (UVIDEC-100-V, Jasco, Tokyo, Japan), with an in-house-modifiedflow cell compartment, operated at 215 nm. The laser-based detection system, used for the studies requiring laser-induced fluorescence detection, consisted of a Series 56X helium-cadmium (He-Cd) laser (Omnichrome, Chino, CA) with an output wavelength of 325 nm and an output power of 30 mW. The laser beam was passed through a mechanical light chopper
(Model 7500, Rofin Ltd., Egham, England) and a focusing lens prior to impinging upon the optical window of the capillary, which was prepared by removing a short segment of the outer polyimide capillary coating. The fluorescent light was collected at a right angle by a 20/0.50 objective lens (Melles Griot, Irvine, CA) and passed through an aperture into an adjustable-wavelengthH-20 monochromator (Instruments SA, Inc., Edison, NJ). Signals were monitored with a R928 photomultiplier tube and amplified with a lock-in amplifier (Model 128A, EG&G Princeton Applied Research, Princeton, NJ). Capillary Electrophoresis Separations, All CE separations were performed in fused-silicacapillaries (Polymicro Technologies, Phoenix, AZ) with inner diameters of 50 Nm, outer diameters of 189 pm, and varying lengths. For some of the separations in this study, the inner walls of the CE capillaries were bonded with a thin layer of linear polyacrylamide, as described in ref 30. All buffer solutions were prepared with distilled water and filtered through 0.2-pm Nylon 66 membrane filters (Alltech Associates, Deerfield, IL). Samples were introduced into the separation capillaries by hydrodynamic f l o ~with , ~ a height differential of 22 cm for the two ends of the capillary. For separations done in uncoated capillaries, the capillary was rinsed sequentially with 0.1 M NaOH, distilled water, and buffer, for approximately 1min each, between successive electrophoretic runs. For separations done in coated capillaries, the capillary was rinsed only with the separation buffer for several minutes between runs. Chemicals. All proteins, buffer components, formic acid, hydrogen peroxide (30%),immobilized chymotrypsin,cyanogen bromide, and benzoin were purchased from Sigma (St. Louis, MO). Immobilized TPCK-trypsin was obtained from Pierce (Rockford, IL). Tributylphosphineand 2-methylaziridinewere of the highest purity available and were obtained from Aldrich Chemical Co. (Milwaukee, WI). Ethylene glycol monomethyl ether (methyl cellosolve),2-mercaptoethanol,and 1-propanolwere from Fisher Scientific Co. (Fairlawn, NJ). Reduction and Alkylation of Disulfide Bonds. A modified procedure of Ruegg and RudingerZ1was employed. Under a nitrogen purge, a 2% (v/v) solution of tributylphosphine in 1propanol was prepared. This solution was stored under nitrogen and used within 3 h of preparation. The protein samples, which ranged in size from 4 to 90 pmol, were first lyophilized in Eppendorf tubes and then dissolved in 2-10 p L of a 1:l mixture of 0.5 M NH4HC03(pH 8.2) and 1-propanol. With a nitrogen stream blowing over the sample solution, tributylphosphine (as the 2 % solution) and 2-methylaziridine were added. The volume of tributylphosphineadded corresponded to at least a 10-fold excess of moles of tributylphosphineover moles of disulfidebonds present in the protein sample. The volume of 2-methylaziridine added corresponded to at least a 40-fold excess of moles of 2-methylaziridine over moles of SH groups present in the reduced protein. Following the addition of the reagents, the sample vial was flushed thoroughly with nitrogen, capped, and allowed to remain at room temperature for 3-4 h. The sample solution was then diluted with distilled water and lyophilized. Oxidation of Disulfide Bonds. The general procedure of HirsZ4was followed. The performic acid reagent was prepared by mixing 95 volumes of 99% formic acid with 5 volumes of 30% hydrogen peroxide. This solution was allowed to stand in a c l w d container at room temperature for 2 h prior to addition to the protein samples. The protein samples (5-10-pmol quantities, lyophilized in Eppendorf tubes) were dissolved in 1-2 pL of a 5:l solution of 99% formic acid and methanol. The performic acid reagent (4-5 pL) was added, and the sample vial was placed in a salt-ice bath at -5 to -10 "C for 2 h. The sample was then diluted with distilled water and lyophilized. Preparation of Tryptic and Chymotryptic Digests. Immobilized trypsin or chymotrypsin, loaded into a small-bore reactor column, was used to prepare the enzymatic digests. A detailed description of the procedure used in preparing and using immobilized trypsin reactor columns can be found in ref 12. The only modification of the previously described procedure was the use of 40 cm X 1 mm i.d. Pyrex tubing for the reactor columns, instead of the 30-cm lengths used previously. All other experimental details were the same.I2 Preparation of Cyanogen Bromide Digests. A concentrated reagent solution was prepared by dissolving 50 mg of cyanogen
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
bromide in 500 gL of 70% formic acid. This solution was added to the protein samples (4-10-pmol quantities, lyophilized in Eppendorf tubes) at levels of approximately 100-fold molar excess of cyanogen bromide over the methionine residues in the protein. The sample vials were sealed, protected from light, and allowed to remain at room temperature for 18 h. The samples were then diluted with distilled water and lyophilized. Derivatization of Arginine-Containing Peptides. The following reagent solutions were prepared for use in the derivatization reaction: (1)0.8 M potassium hydroxide, (2) a solution containing 0.1 M 2-meraptoethanol(which stabilizes the resulting fluorescent product) and 0.2 M sodium sulfite (which helps to suppress blank fluorescence),and (3) 0.08 M benzoin, dissolved in ethylene glycol monomethyl ether (methyl cellosolve). The protein samples (90-95-pmol quantities, lyophilized in Eppendorf tubes) were dissolved in 1 pL of water. To these samples were added 1pL of 0.8 M potassium hydroxide, 1pL of 0.1 M 2-mercaptoethanol/O.2M sodium sulfite, and 1p L of 0.08 M benzoin. The samples were heated in a boiling-water bath for 90 s and then cooled in an ice-water bath. A 1-pL aliquot of the buffer being used for the CE separation was added prior to analysis, which served to adjust the pH of the sample.
RESULTS AND DISCUSSION
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Flgue 1. Reaction schemes for the reduction and awcylation of disulfide bonds. (A) shows the reduction reaction involving the use of tributylphosphine (Bu,P) as the reducing agent. (9)shows the subsequent alkylation reaction, using 2-methylariridlne. (C) is the overall reaction, in which tributylphosphineand 2-methylazirMine are added simultane ously to the protein sample.
The primary goal of this work was to develop a series of methods for peptide mapping which would be suitable for use 1B.52 One of the most attractive features of this combination with extremely small quantities of protein. The first issue of reagents for the reduction and alkylation of disulfide bonds which had to be addressed was the cleavage of disulfide bonds, is the fact that the two reagents can be added simultaneously which are present in many proteins. Failure to break these to the protein, since neither tributylphosphine nor its oxide are reactive toward 2-methylaziridine. Thus, the procedure bonds frequently results in incomplete fragmentation of the protein into peptides. A review of the literature reveals that is simplified and sample handling is minimized. The overall the most commonly used procedures for cleaving disulfide reaction scheme for the simultaneousreduction and alkylation bonds include: (1)denaturation of the protein using a conof disulfide bonds is shown in Figure 1C. centrated solution of urea or guanidine h y d r o c h l ~ r i d e , ~ ~ J ~ J ~ Several more features of this particular reaction makes it (2) dialysis to remove excess salts, (3) reduction of the S-S well-suited for use with extremely small quantities of protein. bonds, with 2-mercaptoethanol or d i t h i o t h r e i t ~ l ,and ~ ~ .(4) ~ In addition to being an effective reducing agent, tributylalkylation of the resulting sulfhydryl groups with iodoacetphosphine also serves as a protein denaturant.21 Therefore, amide or iodoacetic a ~ i d . ' ~ Such > ~ l a protocol is quite suitable the need for pretreatment with a denaturing agent such as for relatively large amounts (i.e. microgram or milligram urea, followed by dialysis, is eliminated. Also, since the requantities) or protein. However, when we attempted to duction reaction with tributylphosphine is not an equilibrium perform similar procedures on a much smaller scale (i.e. nareaction but rather proceeds with a 1:l stoichiometry, there nogram protein quantities), problems of excessive sample loss is not a need to use large excesses of the reducing agent to were encountered. Such problems were attributed to the effect a complete reaction. This is in contrast to the more multistep nature of the procedure, necessitating several well-known disulfide bond reduction reactions involving thiol transfers of the sample among different reaction vessels, and reagents, which are equilibrium reactions and require a fairly the inevitable loss of small amounts of sample with each step. large excess of the reagent to force the reaction to While such small sample lmes may be negligible when dealing However, moderate excesses of both tributylphosphine and with relatively large initial quantities of protein, these losses 2-methylaziridinecan still be tolerated in this procedure, due become increasingly significant as the scale of the reaction to their volatility which makes it possible to remove the excess is reduced. reagents with a simple lyophilization step. In our attempts to develop suitable methoda of dealing with A concern in scaling any type of chemical reaction down disulfide bonds on a microscale, we have departed from the to the low-picomole level, particularly in the area of protein previously described reduction and alkylation procedure in chemistry, is how well the reaction actually occurs at the small favor of another known, but less commonly used, procedure. levels in comparison to larger, more conventional quantities. The reaction involves the use of tributylpho~phine'~-~~ as the We have evaluated our sample treatment and separation reducing agent which initially cleaves the S-S bonds. The procedures at the more conventional levels (Le. nanomole, or reaction scheme for this reduction is shown in Figure lA,19 microgram, quantities of a protein) and compared such results which proceeds under mildly alkaline conditions. In conto those obtained with Bpmol quantities of that protein. junction with this reduction is the alkylation of the active Results of this type of comparison for trypsinogen, a model sulfhydryl groups with 2-methylaziridine. While the use of protein with a molecular weight of 23 700 and six disulfide aziridine for such purposes has been previously r e p ~ r t e d , ~ " ~ ~bridges,= are shown in Figure 2. The reduction and alkylation we have opted to use 2-methylaziridine, due to its somewhat procedure of Figure 1was used, followed by digestion with greater stability and commercial availability. It should be either immobilized trypsin or immobilized chymotrypsin,and pointed out that the use of aziridine for the alkylation will separation by CE with UV detection. Electropherograms A result in the introduction of additional trypsin-susceptible and B represent the tryptic digests of 10 nmol(237 pg) and cleavage sites into the protein, because of the similarity of the 5 pmol (119 mg) of trypsinogen, respectively, while (C)and resulting (aminoethy1)cysteinegroups to lysine.52 We have (D) are the analogous chymotryptic digests. The peak heighta not found this to be the case when 2-methylaziridine is used, of the large and small quantities cannot be compared directly, apparently because of the additional branching and steric as different sensitivity settings of the UV detector were emhindrance from the methyl group. The scheme for the alployed. However, the qualitative comparison of the overall kylation reaction with 2-methylaziridine is shown in Figure peak pattern is the main point of interest here, and a peak-
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Minutes Figure 2. Comparison of tryptic (A and 8 ) and chymotryptic (C and D) digests of trypsinogen, separated by CE. Electropherograms A and C were each obtained from 10 n m d samples of trypsinogen, reduced, alkylated, and enzymatically cleaved as described in the text. Eiectropherograms B and D were each obtained from 5-pmoi samples of trypsinogen, prepared as described in the text. CE separation conditions: buffer, 0.05 M 3-(cyclohexylamino)-l-propanesulfonlc acid (CAPS), pH 9.5, 10% methand; capillary, 50 bm i.d. X 65 cm (50 cm to detector), uncoated: applied voltage, 25 kV; current, 10 PA. by-peak comparison reveals a very close similarity of (A) with (B)and of (C) with (D).This verifies that, from a qualitative standpoint, the procedures used are equally viable at relatively large or small levels, with no apparent difficulties encountered in the scale-down of these reactions. The importance of cleaving the disulfide bonds in a protein prior to enzymatic digestion for peptide mapping purposes is demonstrated in Figure 3 for trypsinogen. When the six disulfide bonds occurring in trypsinogen are allowed to remain intact, the cleavage of this protein by trypsin is incomplete, as can be seen in (A). This is often the case when peptide mapping is done on proteins containing one or more disulfide linkages, which either prevent access of the enzyme to all of the cleavage sites or keep some of the cleaved peptides linked together. We have also found that the peptide maps obtained for proteins with intact disulfide bonds are much less reproducible among repeated preparations. For comparison, the electropherogram in Figure 3B is the peptide map obtained when the disulfide bonds of trypsinogen are reduced and alkylated prior to digestion with trypsin. A more complete digestion is apparent. The reproducibility of any peptide-mapping procedure is of utmost importance, because peptide mapping is largely a comparative technique. That is, the peptide map obtained from a protein of interest is often compared to the peptide map of a known or standard protein which has been treated
Figure 3. Comparison of the tryptic digests of 5-pmoi portions of trvpsinogen wlth (A) intact dlsulffde bonds and (e) reduced and akylated disulffde bonds. Approximately 80 fmoi of each sample was introduced into the separation capillary. CE separation conditions were the same as in Figure 2.
in an identical manner. If the sample treatment or separation protocol is not highly reproducible, the comparisons will be misleading or invalid. Therefore, we have investigated the reproducibility of the previously described sample preparation schemes and subsequent CE separations for peptide mapping purposes. A comparison was made of three separate 5-pmol samples of reduced and alkylated trypsinogen which had been digested with immobilized trypsin and separated by CE. The peptide map patterns compared quite favorably to one another, peak-bpeak. From a statistical standpoint, the percent relative standard deviations (n = 5) of the migration times of three different peaks, occurring in the early, middle, and late portions of the electropherogram, were found to be 0.35, 0.39, and 0.41, respectively. The variations in migration times from run to run can be largely attributed to slight changes in the electroosmotic flow occurring within the uncoated fused-silica capillaries," which is, in turn, the possible result of some adsorption of analyte components to the capillary's inner wall. We tried to minimize any capillary wall variations by following a thorough capillary-rinsing procedure between the individual electrophoretic runs, as described in the Experimental Section. The percent relative standard deviations (n = 5) of the height of the same three peaks were calculated a t 1.21, 1.28, and 1.19. The variations in peak height in our experiments are likely due to slight differences in the individual sample sizes, as well as to the manual hydrodynamic injection method utilized. Although peptide mapping is most often used in a qualitative manner, there are instances when quantitation is desired, and reproducibility of peak heights then becomes significant. A similar reproducibility study was done on the chymotryptic maps of three different 5-pmol portions of trypsinogen, which had been reduced and alkylated, digested with immobilized chymotrypsin, and separated by CE. Chymotrypsin cleaves a protein primarily at the aromatic amino acids, in contrast to trypsin, which cleaves at the lysine and arginine residues. Chymotrypsin is used much less frequently than trypsin for peptide-mapping procedures, apparently because it is known to be less selective in ita fragmentation of prot e i n ~ .However, ~~ in using the immobilized chymotrypsin reactor columns, we have found the reproducibility from sample to sample to be quite good. The patterns of the three electropherograms compared well to one another. The percent relative standard deviations (n = 5 ) of migration times and
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peak heights of three selected peaks were calculated as 0.35, 0.34,0.41, and 1.43,1.20,1.34, respectively. Again, the reasons for the variations from run to run are largely due to the CE process, with some contribution from the sample treatment procedure. We have also investigated the use of cyanogen bromide as the protein cleaving agent. Cyanogen bromide cleaves very specifically a t the carboxyl end of methionine residues&and is widely used for peptide-mapping purposes5Band as a means of fragmenting proteins for subsequent amino acid sequencing.57 Again, our focus was on being able to perform the cyanogen bromide cleaving reaction on a few picomoles of protein which had been previously reduced and alkylated. The scale-down of this particular reaction was straightforward, requiring few modifications from the procedures developed for larger quantities of protein. Cyanogen bromide is typically added to the protein in excess, but fortunately the reagent and reaction byproducts are volatilels and can be easily removed from the sample solution by lyophilization. This fact is especially significant in our work with ultrasmall quantities of protein, where the emphasis is on simplifed procedures and uncontaminated final products. Because of the somewhat infrequent occurrence of methionine residues in most proteins, fragmentation by cyanogen bromide generally results in significantly fewer and larger peptides than the tryptic or chymotryptic digests. For example, trypsinogen contains only two methionine residues, resulting in three fragments upon treatment with cyanogen bromide. In the separation of large peptides by CE, one encounters the same sort of problems as when separating proteins by CE.30933Specifically, the larger the peptide, the greater the tendency toward adsorption to the inner walls of the fused-silica capillary. This adsorption process hampers both the efficiency of the separation (peaks tend to be broad, with noticeable tailing) and the reproducibility of migration times and peak heights (or peak areas) from run to run. To overcome the adsorption problems with cyanogen bromide digests, we have used capillaries coated with linear polyacrylamide,Mwhich signifcantly reduce the adsorption of the larger peptides and can be used over a wide range of buffer pH values. Results of the cyanogen bromide cleavage of 4-pmol samples of trypsinogen are shown in Figure 4, where, again, we can see the differences between attempting to perform the fragmentation on a protein with intact disulfide bonds (A) and the same protein with previously reduced and alkylated disulfide bonds (B). The disulfide bonds in native trypsinogen either prevent the complete cleavage by cyanogen bromide or keep the reaulting fragments linked to one another. The procedures described previously and demonstrated with the model protein, trypsinogen, have also been successfully applied to larger proteins containing more disulfide bonds. Human serum albumin (HSA), a protein with a molecular
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Flgurr 5. Tryptic digests of reduced and alkylated human serum albumin (HSA). (A) was obtained using UV detectlot?at 215 nm, from a bpmol portlon of HSA. Approximately 100 fmol of digested HSA was introduced Into the separation capillary. (B)was obtained uslng leser-inducedfkKwescence~,fo#owlngderhratlratknofdigegted HSA with benroln, an arglninsselecthre reagent. The inltial sample size for (B) was 90 pmol, of which ca. 360 amol was Introduced Into the separation capillary. The off-scale peak occurrlng at ca. 17 mln In (B) Is due to the derhratlzatlon reagent. CE separation conditlone for (A): buffer, 0.05 M 24cyckhexyiamino)ethanes~dfonicacid (CHES), pH 9.7; capillary, 50 pm 1.d. X 55 an (40 cm to detector), coated with ilnear polyacrylamide as described In ref 30; applled voltage, 25 kV; current, 12 pA. Separation conditions for (B): buffer, 0.05 M 34cyclohexylamlnol-1-propanesuifonlc acid (CAPS), pH 9.1. 0.06 M SDS, 10% CH,CN; capillary, 50 pm 1.d. X 70 cm (50 cm to detector), uncoated; applied voltage, 25 kV; current 20 PA.
weight of 66500 and 17 disulfide linkages,% has served as another model protein in this study. The total digestion of reduced and alkylated HSA by trypsin should yield 75 fragments, as determined from the known amino acid sequence.@ Figure 5A shows the CE separation of a tryptic digest of 5 pmol(333 ng) of reduced and alkylated HSA, in which UV detection was utilized. Considering the complexity of this mixture, the resolution attained here is reasonably good. We have used capillaries coated with linear polyacrylamideMfor this separation, which helped to improve the resolution over that obtained with uncoated capillaries. Even with the respectable resolution possible with CE, tryptic maps such as that in Figure 5A can be difficult to interpret, due to the abundance of peaks resulting with UV detection. Therefore, we have investigated an alternative mode of peptide mapping, in which only a certain portion of the peptides in the tryptic dgest are detected. This procedure involves selective derivatization of the arginine-containing peptides with b e n ~ o i n , ~ resulting ' in fluorescent products. Laser-induced fluorescence detection is then utilized in place of W detection. Details of the derivatization reaction with benzoin have been previously described.-' The utility of this procedure for obtaining peptide maps of only the arginine-containing fragments in a tryptic digest is demonstrated here with HSA. Digestion of HSA with trypsin should result in 19 argininecontaining peptides and 5 free arginine residues. Thus,derivatization of this sample with benzoin, followed by
884
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
CE with fluorescence detection, should result in a peptide map of 20 analyte peaks, as opposed to the 75 peaks possible with UV detection. Figure 5B is the electropherogram of HSA which has been reduced, alkylated, digested with trypsin, and derivatized with benzoin. The complexity of the peptide map is substantially reduced, in comparison to (A). The sensitivity of laser-induced fluorescence detection of these derivatives is also improved over UV detection of the underivatized peptides. Approximately 100 fmol of digested HSA was introduced into the separation capillary to obtain electropherogram A, while only 380 am01 of derivatized HSA digest was used to obtain electropherogram B. Ironically, despite the improved sensitivity with fluorescencedetection, the original sample size used for (A) in Figure 5 was smaller than that required for (B) (5 pmol vs 95 pmol of HSA, respectively). This is due to the additional dilution of the sample which occurs with the derivatization reaction. Because the final sample solution is less concentrated following derivatization with benzoin, the original quantity of protein taken must be larger to be able to introduce the requisite amount of sample in the limited injection volumes (Le. 20 nL or less) used for CE. Conversely, with the underivatized sample used with UV detection, the lyophilized protein digest can be dissolved in a minimum quantity of water or buffer, thereby maintaining a higher concentration of the sample solution. The reproducibility of the arginine-selective peptide-mapping routine has also been assessed. With the introduction of an additional sample treatment step (derivatization with benzoin) into the overall peptide-mapping scheme, the ability to obtain comparable electropherograms from multiple samples becomes still more significant. Results from the preparation of three separate portions (95 pmol each) of HSA were compared. The patterns among the three examples were very similar, indicating an adequate reproducibility of the entire sample preparation procedure (reduction and alkylation of disulfide bonds, tryptic digestion, derivatization with benzoin) as well as the CE separation and laser-induced fluorescence detection. The percent relative standard deviations (n = 5) of migration time for three selected peaks were 0.44,0.39, and 0.48,while the percent rsds of peak height for the same three peaks were 1.86,1.78, and 1.83. In comparison to the precision obtained with UV detection, the variation in migration time here is similar, while the variation in peak height is slightly larger. This is attributed to the added sample handling involved with the derivatization reaction, and variations in the extent of derivatization for different samples. To complete the study with HSA, fragmentation by cyanogen bromide was performed. HSA, being a relatively large protein, provides a somewhat more interesting example of cyanogen bromide peptide mapping than the previous case of trypsinogen. With six methionine residua, cleavage of HSA by cyanogen bromide should result in seven fragments,varying in size from 31 to 175 amino acids.5s In Figure 6, we see the results of cyanogen bromide cleavage of 5-pmol portions of HSA with (A) intact disulfide bonds and (B) reduced and alkylated disulfide bonds. Again,the significance of breaking the cystine linkages prior to protein fragmentation is evident. With the S-S bonds intact, only three separate peptides can be discerned, while seven fragments are resolved in the reduced and alkylated sample. These results are in concurrence with other studies on HSA.58 All of the previous peptide-mapping examples have been done on proteins in which the disulfide bonds were reduced and alkylated with tributylphosphine and 2-methylaziridine. While this method of preparing proteins for enzymatic or chemical cleavage has been found to be quite satisfactory at the low-picomole level, we have also investigated the utility of oxidation of the disulfide linkages, again using only a few
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0 5 10 15 Minutes Figure 6. Cyanogen bromide digests of HSA wlth (A) intact dlsuifMe brktges and (B) reduced and akyleted dlsulflde bridges. original sample slzes were 4 pmol, of which ca. 80 fmol was introduced Into the Separation capillary. CE Separation condkions: buffer, 0.05 M giutamine, triethylamine (pH 9.7); capillary, 50 pm i.d. X 55 cm (40 cm to detector), coated wlth linear polyacrylamide as described in ref 30; applied voltage, 25 kV; current, 18 PA. 0
5
10
15
picomoles of protein. Oxidation of S-S bonds with performic acid, while effective and simple, tends to be less popular than reduction and alkylation. The reasons for this stem from the fact that the strong oxidizing conditions necessary to break the cystine linkagea also affect certain other amino acids within the protein. Tryptophan is irreversibly degraded to formylkynurenine and several other products, cysteine is oxidized to cysteic acid, and methionine is oxidized to methionine ~ulfone.'~ These alterations of the amino acids are frequently cited as a drawback to using performic acid oxidation, particularly when the interest is in sequencing the protein or determining its amino acid composition. However, if the primary goal is peptide mapping, such changes to the amino acids are leas significant, providing they are reproduciblefrom sample to sample. The oxidation reaction itself is easily adapted to the small levels we are interested in. Performic acid is typically added to the protein in at least a 10-fold molar exceea and is removed at the completion of the reaction through lyophilization. Thus, the process involves a minimal amount of sample handling, which is desirable for our purposes. We have used the performic acid reaction to oxidize the disulfide bonds in 5-pmol quantities of lysozyme, a model protein with a molecular weight of 14 100 and four disulfide bonds.53 A tryptic map of oxidized lysozyme is shown in Figure 7. The number of peaks obtained here is consistent with the number of trypsin-susceptible sites in the sequence of lysozyme.53 Chymotryptic digestion following these oxidation reactions is not recommended, because tryptophan residues (one of the primary cleavage sites of chymotrypsin) are destroyed in the oxidation procedure. Similarly, with the oxidation of methionine to methionine sulfone, cleavage of the oxidized protein by cyanogen bromide is precluded. Thus, digestion by trypsin is the preferred option for peptide mapping following oxidation of disulfide bonds.
CONCLUSIONS A variety of sample treatment methods for use in the peptide mapping of cystine-containing proteins have been presented. The emphasis throughout this study has been on the use of each method with very small quantities of protein. With the exception of the fluorescent derivatization reaction of tryptic digests, all procedures have been successfully carried out on 4-5-pmol amounts of different model proteins. The key to working with these small sample sizes has been to minimize sample handling, while still performing all necessary reactions to prepare the proteins for peptide mapping.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
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a complement to the more common UV detection of underivatized samples, to gain additional information on complicated peptide mixtures.
ACKNOWLEDGMENT This research was supported by Grant No. GM 24349 from the National Institute of General Medical Sciences, U.S. Department of Human Health and Services.
REFERENCES
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Minutes Flgure 7. Assessment of reproducibility of tryptic mapping of oxidized lysozyme. Spectra (top to bottom) represent three separate sample preparations of 5 pmoi each. Approximately 100 fmol of digested lysozyme was introduced into the separation capillary. CE separation conditions: buffer, 0.05 M sodium phosphate (pH 2.3);capillary, 50 pm 1.d. X 65 cm (50 cm to detector), uncoated: applied voltage, 27 kV; current, 20 PA.
The use of tributylphosphine and 2-methylaziridine for reduction and alkylation of disulfide bonds represent a departure from the more conventional procedures, but these reagents exhibit particular advantages when working a t the low-picomole level. Oxidation of disulfide linkages has also proven to be a viable method a t these scaled-down levels. Although the oxidation reaction limits the subsequent protein digestion methods (due to the alteration of certain amino acids), it is still an option which may be well suited for particular applications. The use of immobilized proteolytic enzymes for digesting the proteins is a seemingly small, but critical, modification of the conventional procedures developed for use with larger quantities of protein and has allowed us to reproducibly digest a few picomoles of protein. Cyanogen bromide fragmentations of proteins are wide!y practiced, but we have demonstrated their applicability to protein quantities that are 3-6 orders of magnitude smaller than normally utilized. Highly efficient CE separations of the larger cyanogen bromide-generated peptides have necessitated the use of wall-coated capillaries, previously developed for protein separations, to minimize adsorption of the large peptides to the capillary’s inner wall. While W absorbance is the most commonly used detection mode for peptide-mapping applications, we have demonstrated an alternative routine using laser-induced fluorescence detection. The selective reaction of benzoin with arginine residues allows one to obtain partial peptide maps of only the arginine-containingpeptides. A close match of the excitation maximum of such derivatives with the output of a 325-nm He-Cd laser results in attomole detection limits. This alternative mode of peptide mapping may be used alone or as
Garnick, R. L.; Solli, N. J.; Papa, P. A. Anal. Chem. 1988, 60, 2546-2557. Regnler, F. E. LC-GC 1987. 5 , 392-398. Regnier, F. E. LC-GC 1907. 5, 472-474. Vensel, W.; Fujita, V.; Tarr, G.;Margolish, E.; Kayser, H. J. Chromafwr. 1983, 266, 491-500. Stone, K.; Williams, K. J. Chromafogr. 1986, 359, 203-212. Haefner-Gormley, L.; Poludniak, N. H.; Wetlaufer, D. B. J. ChromafOgr. 1981, 274. 185-196. Huberman, A.; Aguilar, M. 8. J. Chromatogr. 1988, 443, 337-342. Edeiman. 0. M.; Gail, W. E.; Waxdal. M. J.; Konigsberg, W. H. Bbctmmlshy 1968, 7 , 1950-1958. Canfieid, R. E.; Anfinsen, C. B. J. Bioi. Chem. 1963, 236, 2684-2690. Kalghatgi, K.; Horvath, C. J. Chromafogr. 1988, 443, 343-354. Simpson. R. J.; Morkz, R. L.; Begg, 0. S.; Rubira, M. R.; Nice, E. C. Anal. Blochem. 1989, 177. 221-236. Cobb, K. A.; Novotny, M. Anal. Chem. 1989. 61, 2226-2231. Lundblad, R. L.; Noyes, C. M. Chemical Reagents for Profeh ModltYcation; CRC Press, Inc.: Boca Raton, FL, 1984; Voi. I,Chapter 7. Means, G. E.; Feeney, R. E. Chemlcel hbdlficetbn of Proteins; HOC den-Day, Inc.: San Francisco, CA, 1971; Chapter 8. Smyth. D. 0. Methods Enzymol. 1967, 7 7 , 214-231. Stone. K. L.; LoPrestl, M. B.; Williams, N. D.;Crawford, J. M.; DeAngelis, R.; Williams, K. R. I n Techniques In proteln Chemistry; Hugii, T. E., Ed.; Academic Press, Inc.: San Diego, CA, 1989; pp 377-391. Humphrey, R. E.; Potter, J. L. Anal. Chem. 1965, 37, 164-165. Sweetman, 8. J.; Maclaren, J. A. Aust. J. Chem. 1968, 19, 2347-2354. Fontana, A.; Gross, E. I n Practical Protein Chemistry; Darbre, A,, Ed.; John Wlley 8 Sons: New York, 1986; pp 67-120. Maciaren, J. A.; Sweetman, B. J. A M . J. Chem. 1966, 19, 2355-2360. Ruegg, U. T.; Rudinger. J. Methods Enzynwl. 1977, 47, 111-116. Raitery, M. A.; Cole, R. D. J. Bbl. Chem. 1966, 241, 3457-3461. Tsung, C. M.; FraenkelConrat, H. Blochemistry 1966. 5 , 2061-2067. Hirs, C. H. W. Methods Enzymol. 1967, 7 1 , 197-199. Firestone, M. A.; Michaud. J. P.; Carter, R. N.; Thormann, W. J. Chromafogf. 1987, 407, 363. McCormick, R. M. Anal. Chem. 1988, 60, 2322-2328. (;rossman. P. D.: Wilson, K. J.; Petrb, 0.; Lauer, H. H. Anal. Bkche” 1988. 173. 265-270. Gros&”a,’P. D.; Coiburn, J. C.; Lauer, H. H.; Nieisen, R. 0.; Riggln, R. M.; Sittampalam, G. S.; Rickard, E. C. Anal. Chem. 1989, 61, 1186-1194. Stover, F. S.; Haymore, B. L.; McBeath, R. J. J. Chromafogr. 1989, 470, 241-250. Cobb, K. A.; Dolnik, V.; Novotny, M. Anal. Chem. 1990, 6 2 , 2478-2483. Lauer, H. H.; McManlgill, D. Anal. Chem. 1986, 5 8 , 166-170. Bruin, 0. J.; Chang, J. P.; Kuhlman, R. H.; Zegers, K.; Kraak, J. C.; Poppe, H. J. Chfomrogr. 1989, 471. 429-436. Towns, J. K.; Regnier, F. E. Anal. Chem. 1991, 6 3 , 1126-1132. Cohen. A. S.; Najarian, D. R.; Pauius, A.; Guttman, A.; Smith, J. A.; Karger. E. L. R o c . Mefl. Aced. Scl. U.S.A. 1988. 85, 9660-9663. W e n . A. S.; Terabe, S.; Smith, J. A.; Karger, 8. L. Anal. Chem. 1987. - - - - , 59. 1021-1027. -Cohen, A. S.; Naja;&, D.; Smith, J. A.; Karger, 8. L. J. Chromafogr. 1988. 458. 323-333. Liu, J.; Shirota, 0.; Wiesier, D.; Novotny, M. Roc. Nefl. Aced. Sci. U.S.A. 1991, 88, 2302-2306. Honda, S.; Iwase, S.; Makino, A.; Fujiwara, A. Anal. Biochem. 1989, 176, 72-77. Hoffstetter-Kuhn, S.; Paulus, A.; Gassmann, E.; Widmer, H. M. Anal. Chem. 1991, 6 3 , 1541-1547. Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromafogr. 1983, 264. 385-392. Gozei. P.: Gassmann. E.; Michelson, H.; Zare. R. N. Anal. Chem. 1987, 5 9 , 44. Huana, X.; Gordon, M. J.; Zare, R. N. J. Chromafogr. 1988, 425, 385-390. Llu., J.: - . Cobb. K. A,: Novotnv. M. J. Chromatoor. 1989. 468. 55-65. Burton, D. E.: Sepaniak, M. Maskarlnec, M. F. J. Chromfogr. Scl. 1986, 2 4 , 347-351. Cobb, K. A.; Novotny, M. V. Anal. Blochem., in press. Kai. M.; Ohkura, Y. Trends Anal. Chem. 1987, 6 , 116-120. Kai. M.: Mlvazaki. T.: Yamaauchi. M.: Ohkura. Y. J. Chromatow. 1963, 266,-417-424. Rose, D. J.; Jorgenson, J. W. Anal. Chem. 1988, 60, 642-648. Bennett, J. C. Methods Enzymol. 1987, 1 7 , 211-213. Cieiand, W. W. Blochemlsby 1964. 3 , 480-482. Hirs, C. H. W. MethodsEnzymoI. 1967, 7 1 . 199-203.
--.
(43) (44) (45) (46) 147) .
I
(48) (49) (50) (51)
~
i;
-
Am/. Chem. 1992, 64, 886-891
880
(52) Means, 0. E.; F e e ~ y R. , E. chdmlccrl AWbkaths of Proteins: HOC dm-Day, Inc.: Sen Francisco, CA, 1971;Chapter 6. (53) Croft, L. R. Hendbwlr of Protek, Sequence Ane!~sb;John Wiley & Sons: Chlchester. U.K.. 1980. (54) Lambert. W. J i -ik&ton, D. L. AMI. m m . 1990, 62, 1585-1587. (55) QOSS, E. MeEMymd. 1967, 1 1 , 238-255. (56) oloss, E.; Witkop, J. J . Bld. Chem. 1962, 237, 1856.
(57) Simpson, R. J.; Nice, E. C. Biochem. Int. 1984, 8, 787-791. (58) Meloun, B.;Moravek, L.; Kostka, V. FEBS Lett. 1975, 58, 134-137.
RECEmD
for review September 20,1991.Accepted January
9,1992.
Effect of Direct Control of Electroosmosis. on Peptide and Protein Separations in Capillary Electrophoresis Chin-Tiao Wu, Teresa Lopes, Bhisma Patel, and Cheng 5.Lee* Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County Campus, Baltimore, Maryland 21228
The separations of peptide and proteln dxtures In capMary zone ehctrophorda (CZE) at various dutlon condltlow were rtudkd wlth the dlrect control of eMrooanorls. The f potential at the crqmoulcrrplrry hterface and the remHed eiecttoormosk In the presence of an dectrlc fkld were dC redly controlled by using an addttbnal ehctrk Ikld applied from outrld. of the capillary. The contmhd electrmotlc IkWcr(hcledd~mlgraknthtW8ndzom,nroMknol~
and proteln mlxtures. The changes In the magnltude and polarlty of the potential cawed the varlow degrees of peptide and protein aWnpikm onto the ccrplllary through the dectrootatk Interactlons. The wparatbn effkkncks of peptide and protdn mlxtures were enhanced d m to the reductknkrp@kJe(Md~0hla d ” a t d l s a p b y Wall. The direct manlpulatlonr of the separation etflokncy and resoluth of peptide and protdn mixtures In CZE were demonstrated by sbnply controlling the f potential and the doct r m o t l c flow wlth the appllcatkn of an external electric Ikld.
INTRODUCTION With the application of the current monitoring method’ and the UV marker method,2 we have recently proposed and demonstrated the direct control of electroosmosis in capillary zone electrophoresis (CZE) by using an additional electric field applied from outside of the capillary.” The potential gradient between the external and internal electric potentials is perpendicular a c r m the capillary wall and controls the polarity and magnitude of the 5 potential on the interior surface of the capillary wall. Because the direction and flow rate of e1ect”ceis are dependent upon the polarity and magnitude of the ( potential,B the electroosmotic flow can therefore be directly manipulated by varying the external electric field. In CZE, the electroosmotic flow affectsthe amount of time a solute resides in the capillary, and in this sense both the separation efficiency and resolution are related to the W o n and flow rate of e l e c t m o e i s . 7 In thisstudy, the separations of peptide and protein mixtures in CZE with the direct control of electroosmosis are described. The effect of such control on the separation efficiency and resolution of peptide and protein mixtures is discueeed. The direct manipulation of the separation efficiency and resolution in CZE can be easily obtained by simply changing the external electric field. *Towhom all correspondenceshould be addressed. 0003-2700/92/0364-0886$03.00/0
EXPERIMENTAL SECTION The experimental setup for directly controlling the electroosmotic flow in capillary electrophoresisby using an additional electric field applied from outside the capillary has been described in detail in the previous study? Peptide and protein samples were introduced into the inner capillary by using the electromigration injection method? No external electric field was used during the injection period. For peptide and protein separations at various applied potential gradients, the cathode end of the inner electric field was always in reservoir 4,the UV detector end. The electrophoretic migration of peptide and protein mixtures at various solution conditions examined in this study was always toward reservoir 4,the cathode end of the inner electric field. A nonionized molecule, dimethyl sulfoxide, in the solution mixture was used as the e l e c t m o t i c flow marker. The change14 in the diredion and flow rate of electrooemosis with the application of an external electric field were measured. The flow rate of electroosmosiswas assigned as positive when the direction of flow was toward the cathode end of the inner electric field. The direction of electroosmosis was toward the anode end when the { potential at the inner capillary/inner solution interface was changed from negative to positive6with the application of strong positive potential gradients across the inner capillary wall. In order to measure the negative value of electroosmotic mobility by using dimethyl sulfoxide as the flow rate marker, the cathode end of the inner electric field was temporarily changed from reservoir 4 to reservoir 1. The sodium phosphate buffer, dimethyl sulfoxide, hydrochloric acid, and all peptides and proteins were purchased from Sigma (St. Louis, MO). The pH of the buffer solution was adjusted with the application of 0.1 N hydrochloric acid. The separations of peptide mixtures in a 23-cm-long inner capillary with a 50-pm i.d. and 150-rm 0.d. were studied. All peptides except the adrenocorticotropichormone fragment 4-10were dissolved in 10 mM phosphate buffer of pH 2.7 at concentrationsof approximately 200 pg/mL. The adrenocorticotropichormone fragment 4-10was dissolved in the same buffer at a concentrationof approximately 40 Peptides were injected by using 1-kV inner electric potential for 10 s without the application of an external electric field. A constant inner electric field equal to 239 V/cm (5.5 kV over 23-cm-long inner capillary) was then applied for electrophoresis. The separation distance between reservoir 1 and the UV detector was 14.5 cm.
RESULTS AND DISCUSSION Peptide Separations. The experimental result for peptide separation in the absence of an external electric field was shown in Figure 1. The elution order was Lys-Trp-Lys, thymopoietin I1 fragment, adrenocorticotropic hormone fragment 4-10,bradykinin, and human angiotensin 11. The theoretical charges on the peptides were calculated by using Skoog and Wichman’s model? The parameter, charge/(mo0 1992 American Chemical Society
