AM/. Chem. 1994,66, 2382-2389
Postcolumn Radtarwflde Detection of Low-Energy @ Emitters in capillary Cledrophurds Scott T r a , Vwns Toma,t and Jonathan V. $woodor’ Lbpartment of Chembtry, Univmfty of Il~Ytwis,M a n e , Ilkhois 61807
A postcolumn radionuclide detection system for capiuVr electropboresrs (CE) is described. Eluant from an ekctropboresis capillary is directed onto a peptide M.diss “e that has ban preriolrply coated with a solid scintillator. ”be wmbrane is moved in a prese&cted pattern rehtive to the fixed caplllur outlet duriBg ckctropbonds. Light emission
fromscintllhtioaisiomguloatoac~kriee(CCD) wing a series of 35-mm camera kms. Detection of two lowenergy @- emitters (3% and %) not p m i o d y nported for capillary electropbonsis is de”tr8ted Tk SCpMtioa efficiencies are similar to tbose obcriaed witb opline UV detection. Tbe response for %4beled metbkdae b liacu (S = 0.996) from 66 .mol to 11 fmol. Detection h i t s are 88 zmol(0.03 Bq) for 3Wabdedrarlytes, 17 am01 (0.94 Bq) for %-labeled mrlytes, and 8 fmol(8.5 Bq) for %-labeled arlytes. Radioisotopes have been used as an analytical tool in biochemical applications for a number of years. This method of labeling and detection offers attomole sensitivity and unprecedented selectivity. Because of these features, radionuclidedetection is extensivelyused for a variety of separation techniques such as liquid chromatography and slab gel electrophoresis.1*2 The ability to selectively label particular compounds in matrices such as a living cell allows extremely complex samples to be investigated using conventional separation methods. In addition, the ability to label a small fraction of a particular compound can be important. For example, labeling newly synthesized peptides in a cell (by introducing radiolabeled precursor for a short period) allows the dynamics of peptide synthesis and transport to be studied in ways that other detection schemesdo not a l l ~ w Because .~~~ the analysis of small amounts of complex mixtures of biologically important molecules is among the strong points of capillary electrophoresis,a natural progressionis to combine radionuclide detection with capillary electrophoresis. Kaniansky and co-workers first demonstrated on-column detection of the radionuclides 32P, W , and 99Tc for capillary isotachoph~resis.~.~ Their system consisted of 300 pm i.d. capillaries and a plastic scintillator/photomultiplier tube + R a e n t address: Eli Lilly and Co.,I a d i ~ p o l i r ,IN 46254. ( I ) Andrcw,A.T. Elecrrqphoreds, 2ndd.;OxfordScicnaPuMiatiom: Oxford, 1990, Chapter 2. (2) Rcich. A. R.; Lucar-Rcich. S.;Parva, H. Prog. HPLC 198&3, 1-10. (3) Lloyd. P. E.; Schackr. S.;K u p f e r ” , 1.; W k , K.Proc. Notl. Acad. Sci. U.S.A. 198683.9794-9798. (4) Church, P. J.; Lloyd, P. E. 1.Nnvorc&ncc 1991. 1 1 , 618-625. ( 5 ) Kaniamky. D.; Rajec, P.; svec, A.; Ha&, P.; Madlek. F. 1.Chromarogr. 198% 258, 238-243. (6) Kaniamky, D.; Rajec, P.; svec, A.; Mar& J.; Kovd. M.;LO&, M.; Fnnko. Saband, G. J. Radioanal. Nucl. Chcm. 1989, 129, 305-25.
s.;
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Amlyflcel M t t y , Vd. 66, No. 14, July 15, 1994
combination to detect /3- particles. They reported a detector efficiency of 15% and a 6 pmol (16 Bq) LOD for 1% using a large (210 nL) detector cell. Altria reported CE separations and detection of radiolabeled pharmaceuticals containing q c , a P emitter with a 6-h half-life? Pentoney and Zare demonstrated 32Pdetection in an on-line capillary electrophoresis ~ y s t e mthey ; ~ ~reported ~ three different approaches, including the use of coincidencedetection with nearly a 100% detector efficiency for 32P. In their systems, a 100 pm i.d. capillary and relatively large injections of between 60 and 80 nL were used. With this approach, the reported LOD was approximately 40 amol during a standard CE run. Several additional reports have investigated the detection of energetic 8- and y emitters,loJ1and severalon-column approacheshave been commercialized.12J3 However, almost all previous work has used on-column detection and is not suitable for detection of two of the most important nuclides for biological work-JH and 3%. One of our long-term goals is to detect 3sS-labeled methionine and cysteine containing peptides from subsections of a cell, and we need the ability to detect these weak @- emitters. The inherent problem with these radionuclides is that a /3- particle has insufficient energy to penetrate the capillary wall.14 This leaves two approaches to detect these emitters: placing a scintillator in the running buffer or on the capillary wall, or using postcolumn detection. In this report, we describe a postcolumn membrane/scintillator that allows peptides and other relatively small molecules labeled with 32P,’H,or 35S to be detected. In radionuclide detection, the longer one can observe the sample, the lower the limit of detection. In capillary electrophoresis, typical peak widths are only several seconds wide, and so only a several-second observation is possible without limiting separation efficiency. In order to detect a radiolabeled analyte, the probability of observinga peak must be high-in other words, several decays must be observed, even in background-free situations.ls As a 3-am01 sample of (7) Altria, K. D.; Simpwn, C. F.; Barij, A.; Th&ld, A. Roontcd at the 1988 Pittsburgh Conteteaa, Ncw 0 r l ~ February ~ , 1988.Paper no. 642. (8) Pentmy. S.L.; ZUC,R. N.; Quint, J. F. AMI. Ch” 1969.61, 164247. (9) Pcat0ney.S. L.; hre,R. N.;Quint, J. F. Ana1yticalB&rcchdogy: Capillary E l c c t r o p ~ d s a n d C h r o m a t o g r a p Horvlth, h~ C.. Nikelly, J. G., Edr.;ACS Sympaium Scria No. 434; M a n Chemical Society: Washington DC, 1990; pp 60-89. (IO) Watcrber&G.;Lundquirt,H.;Kihr,F.;Longrtrom,B.J. Chromufolp.1993, 25. 319-325. (1 1) Gordon, J. S.;Vuile, S.;Hadctt, T.; Squillante. M.IEEE Trow. N w l . Sci. 1993.40, 116244. (12) Ism, lac. Product Guide, I I U ~ ~ I ~ forCChromatography IUS and Elcctrophwrsir; Lincoln, NE, 1993. (13) Raytest USA. Rndiodcfccrionin CE, Wilmington, DE, 1992. (14) Knoll, G. F. Radiation Dctecrion and Mcrrrurcmcnt, 2nd d.;John Wiley: New YorL 1989.
ooo3-2700/94/03862382so4.M)Io Q 1994 Amerlcen Chemical Society
32P has an activity of 1 Bq (one decay/s), the best possible LODs are on the order of 8 amol for a I-s observation time. In previous work by Pentoney,g the detection efficiency of their most sensitive coincidence detector is nearly 100%. Therefore, further improvements are difficult using an online approach. One of the few methods available to further improve LODs is to observe the analyte for a longer time. Pentoney and co-workers increased the observation time by lowering the applied potential during the transit of a peak of interest past the detector. Using the flow programming approach, they demonstrate an improvement in LODs of more than an order of magnitude. As they note, one must know precisely when the peak of interest will reach the detector. Using the flow programming method for samples containing a number of analytes eluting over a wide time range can be problematic. An advantage of postcolumn detection is that it isolates detection conditions from the separation conditions-the separation can be optimized independently of the observation of the membrane. Even the low efficiency (typically only a few percent) of photographic film used in autoradiography can produce superior results to coincidence detection because of the long observation times possible. Using a postcolumn detector requires collecting effluent fractions. The collection of CE effluent is complicated by the fact that the end of the capillarymust be electrically connected to complete the electrophoresiscircuit. Membrane fraction collection in CE was first demonstrated by Huang and Zare using a CE system grounded before the outlet with an oncolumn frit.16 The outlet end of the capillary made contact with a moving piece of filter paper, and fluorescentlylabeled amino acids were visualized and detected using photographic film. HjertCn and co-workersl7 and Carson and colleaguesls demonstrated improved versions in which a capillary touched a moving, grounded membrane to complete the circuit. In both systems, a circular membrane was rotated to produce a phonograph-like recording of the capillary effluent. In the report by Hjertbn, a sampleof radioactive iodine labeled human growth hormone was separated and eluted onto a membrane. The membrane was then cut into 20 sections and the pieces counted in a y counter. In these previous reports, the separation efficiency for the protein separationswas less than 20 000 plates; whether this was due to heterogeneity in the samples, interactions of the analyte with the capillary wall, or zone spreading caused by the membrane fraction collection was not determined. We also use a membrane to collect the capillary eluant. A variety of membranes were tested for this application; although a number of protein binding membranes are available, few are available which bind smaller molecules such as small peptides. We are using a prototype membrane (Immobilon Psq/CD Experimental) from Millipore Corp. with a high surface area and a hydrophilic cationicpolymer (which should bind the carboxylic acid group of the peptides). Malcolm Pluskal of Milliporereports that it quantitatively binds small peptides.I9 (15) Currie, L. A. Anal. Chem. 1968,40, 586-93. (16) Huang, X.; Zare, R. N. J. Chromorogr. 1992,516, 185-89. (17) Eriksson, K.-0.; Palm, A.; HjertCn, S. Anal. Biochem. 1992, 201, 211-15. (18) Cheng, Y.-F.; Fuchs, M.;Andrews,D.;Carson, W . J . Chromarogr.1992,608, 109-1 6.
Table 1. Phyrlcal Prop.rtkr of Selected RadlormclW half-life mode emission mol/Bq nuclide (dl ofdecay (MeV) (amol)
77Br 3*P 1251
35s
3H 1 4 c
2.4 14.2 51 87 4500 2.03 X 106
B BPY
; 88-
0.361 1.71 0.149 0.167 0.019 0.156
0.49 3 12 18 930 4.30 X 106
In our case, the membrane binding properties are complicated by the desire to pretreat the membrane with a scintillator such as PPO (2,5-diphenyloxazole) and a wavelength shifter (perylene). Using PPO and perylene, we can and 3H-containinganalytes. The membrane detect 32P-,35S-, has a series of glowing analyte bands after the CE run, with the light emission from each analyte in proportion to the amount of radionuclidepresent. However, the PPO/perylene scintillator mix is hydrophobic and changes the ionic binding properties of the membrane. Another complication is the requirement for an efficient CE separation, as we wish to separate complex mixtures of similar peptides. Thus, the choice of membrane/scintillator is extremely important for the success of this radionuclide detection approach. In preliminary work, a nonoptimized membrane/scintillator system was used, and the resulting separation efficiency was poor, most likely due to postcolumn band broadening20 We currently use the membrane dry, electrically connect the capillary outlet to ground, and then raster the membrane lightly across the stationary capillary. Using this approach, we obtain up to 100 000 theoreticalplates for peptides and amino acids with improved detection limits for radionuclides. Once the bands are deposited onto the membrane and dried, the next step is to image the membrane and quantitatively measure the light emission from each band. In order to accomplish this, we use a series of 35-mm camera lenses coupled to a cooled, scientificcharge-coupled device (CCD). CCD detectors are now used for a number of measurements in spe~troscopy~l-~~ and are well suited to this detection system. The characteristicsthat make CCDs well suited for measuring photon emission from small points of light in the night sky, low dark current, and read noise also make them the detector of choice for measuring light emission from the membrane. With membrane observation times between 1 and 2 h, the LODs for single-labeled compounds are 88 zmol (0.03 Bq) for 32P,17 amol (0.94 Bq) for 35S,and 8 fmol (8.5 Bq) for 3H. The reasons for the great variation in LODs for the different nuclides are twofold: the lifetimeof the radionuclide and the energy of the B particle. Table 1 lists the activity and lifetimesfor several commonly used radionuclides. A further advantageof this postcolumn method is that after the detection procedure, the membrane is still intact, and the measurement (19) Malcolm Plush1 (Millipore/Waters). Private communication, July 1993. (20) Sweedler, J. V.; Fuller, R.; Tracht, S.;Timperman, A.; Toma, V.; Khatib, K. J . Microcolumn Sep. 1993, 5, 403-412. (21) Sweedlcr, J. V.; Bilhorn, R. B.; Epperson, P. M.; Sim, G. R.; Denton, M. B. Anal. Chem. 1988, 60, 327A-335A. (22) S w d l e r , J. V. Crir. Rev. Anal. Chem. 1993, 24, 59-98. (23) Charge TransferDevieesinChemis?ry,Swdler. J.V.,Ratzlaff,K. L.,Denton, M. B., Eds.; VCH: New York, 1994.
Analytical Chemistty, Vol. 66,No. 14, Ju& 15, 1994
2585
EXPERIMENTAL SECTION CE System. The radiochemical detection capillary electrophoresis system is shown in Figure 1. With the exception of the membrane fraction collection system, the CE system is similar to previous~ystems.2~ The entiresystem is contained within a Plexiglas box with interlocks. A 75 pm i.d. fused silica capillary (Polymicro, Phoenix, AZ) was used for all experiments. All injections use gravity injection with a 9-cm height displacement for 10s; the injectionvolume is calculated to be 5 nL under these conditions.25 A 20-kV potential was
applied to the injection end for all separations using a Bertan high-voltage power supply (Model 230, Bertan, Hicksville, NY). In order to facilitate membrane fraction collection, a silver electrode is fabricated on the outside of the outlet end of the capillary. The electrodeis constructed by painting conductive silver paint (GC Electronics; Rockford, IL; Part No. 22-202) on the last centimeter of the capillary. The coating is heated to 125 “C for 2 h to improve durability. The electrical connection to the power supply, shown in the detail of Figure 1, is maintained with a nonconductive collar that holds a conductive screw against the electrode coating. Connection to ground is made with an alligator clip attached to the screw. This arrangementallowsthe CE circuit to be maintained with no electrolyte at the outlet end (except that provided by electroosmotic flow). Such painted electrodeshave been used daily for several months with no degradation in performance. CE separation conditions and parameters are optimized using a Waters Quanta 4000 CE system with UV detection. Nonderivatized amino acids and peptides are detected using the standard Waters detector set at 185nm. In order to allow comparisons of the membrane collection system with this oncolumn detection system, the capillary diameter and length, the applied voltage, and injection conditions are kept nearly equivalent in both systems. Membrane FractionCollection. The eluant is directed onto a membrane that has been impregnated with a solid scintillate by physicalcontact of the end of thecapillary. The membrane is moved in a preselected pattern relative to the fixed outlet of the capillary during the CE separation. The membrane is moved by fastening the membrane onto an XY translation stage with the stage position controlled using linear translation motors and a manipulator controller from Newport (Model 860CM2 controller, Model 860SA motors, and 230stages, Newport Research Corp., Irvine, CA). The membrane movement is programmed using Lab Windows (Version 2.2.1 from National Instruments; Austin, TX) driving a Lab PC card (National Instruments; Austin, TX) in a Northgate 80486 33-MHz IBM AT compatible computer, which also controls the electrophoresis power supply. Highly efficient light collection optics are available for small-membrane areas, leading to low LODs; on the other hand, too small a membrane can reduce separation efficiency. As a compromise, we use a 2.5 cm by 2.5 cm membrane to record a 16-min capillary output trace. Figure 2 shows an image of a fraction collection membrane when the running electrolytecontains a small amount of 35S-labeledmethionine and the membrane has been pretreated with a scintillator, thus allowing the capillary trace to be visualized. The membrane movement is 2.5 cm/min for all separations. The width of the trace is approximately 0.6 mm for 3H- and 3sSlabeled amino acids and peptides, and slightly wider for 32P analytes. A number of membranes and scintillator materials have been evaluated. The membrane performs two different tasks-binding the analytes and emitting photons. Beckman’s XtalScint membrane, which uses a yttrium silicatescintillator coated onto a fiber support back, has the highest light emission
(24) Sweadler, J. V.; Shear, J. B.; Fishman, H. A.; are,R. N.; Scheller, R. H. Anal. Chem. 1991,63,496-502.
(25) Jandik, P.; Bonn, G. Capillaty Electrophoresis of Small Molecules and low, VCH: New York, 1993; Chapter 3.
polylmlde coatlng /
caplllary
‘L
, , ,/,,
II
I I
II
sl7eve /
\
contact
I
Capillary Holder
Caplllary Column
‘Hlgh Voltage
8
Power Supply
4...............*. ....*. ......... *
X-Y Motorized Translation Stage
-
/
/
/
Reversed Camera Lens
Normal Camera Lens
Collectton Membrane with Sclntlllator
Flgure 1. (A, Top) Schematicdlagam of the capillaryelectrophoresis/ membranefraction collector system. The detail shows the end-column electrical connection that completes the electrophoresis circuit. (B, Bottom)Membraneimagingsystem. Two 3 5 ” camera lenses image the membrane from the 2.5 cm by 2.5 cm membrane containing the eluant from the CE run onto the 1 cm by 1 cm CCD array.
can be repeated many times to decrease the LODs (with a corresponding increase in analysis time). In addition, many analytes are available with multiple radiolabels, which can further lower the obtainable LODs.
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2384 AmmlChemisW, Vo!. 66, No. 14, Ju& 15, 1994
m
I
n
-
.- ---
--
----- ,..L
-2. Imegeofthemembranewhenthebuffercontainsradidabeled methionine, showing the path of the outlet end of the capillary. Each horizontal line represents 1 mln.
of any tested scintillator/membrane. Although the scintillation response is excellent, the number of theoretical plates for a peptide separation has never been more than 1000.20In addition, the underlying fibrous support is easily torn by the motion of the capillary. Several membranes designed for blotting proteins and peptides from slab gels have been evaluated. We find that scintillation response and peptide binding is highest with Immobilon Psq/CD (experimental) from Millipore coated with PPO. Binding and response are related in that more tightly bound peptides generally occupy a smaller area of the membraneand light from scintillationis therefore concentrated in a smaller area on the CCD. Although the Millipore membrane also produces broad peaks when prewetted with running buffer (as is commonly done), it produces much narrower peaks than any other tested membrane when used dry and moved lightly across the capillary end. The use of dry membranes is possible in this system,as opposed to previous membrane collection systems, because of the end-column grounding method. Scintillation efficiency of the membrane is optimized with compounds commonly used for liquid scintillation counting. After the screening of several compounds, PPO has been selected as thescintillator. However, PPO has a light emission below 400 nm at a wavelength that the CCD has poor quantum efficiency. Because of the much higher quantum efficiency of the CCD at longer wavelengths, perylene, with an emission in the 45&500-nm region, is used as a wavelength shifter. The optimum membrane soak solution is 0.1 g of PPO and 0.5 mg of perylene per mL of diethyl ether. Although ether is chosen as the solvent because it solubilizes the scintillators and does not degrade the membrane coating significantly, a pure ether treatment extracts some membrane as it reduces the membrane weight by -4%. The membrane is soaked in the ether solution for 5 min and in allowed to dry for 15 min at room temperaturebefore use. Membranes are treated and used the same day.
Detection. The membrane is imaged onto the CCD using two standard Nikon 35-mm camera lens, as shown in Figure 1B. The lenses provide approximately 2.7: 1 image reduction, so that a 2.5-cm membrane can be imaged by a 1-cm CCD. The camera lens near the membrane, a Nikon 135-mm f2.0 lens, is used in reverse, and the lens near the CCD, a Nikon 50-mm f1.2 lens, is operated in the normal direction. Both lenses are focused to infinity and set to maximum aperture. The reversed lens collects light from the membrane and collimates it, while the second lens focuses the collimated light onto the CCD. The overall collection efficiency of the system is equivalent to 4 2 . 5 and represents a compromise between light collection efficiency from a point on the membrane and the membrane area. A liquid nitrogen cooled 16-bit CCD is used to detect the light emission from the membrane. The CCD is a PM512 controlled with a CH260 camera controller (Photometrics Ltd., Tucson, AZ). It has a low read noise and dark count rate, allowingmultiple-hourintegrationtimes of the membrane emission. The CCD is controlled using PMIS 2.0 software, also from Photometrics. CCD readout is a very flexible process, with variable analog gain, binning, bias procedures, and a variety of postacquisition image processes available to improve performance. For this work, the binning is set to 4 serial by 4 parallel, resulting in 128 by 128 point images of the membrane. Even with a read noise floor of approximately 10 e-, this system is usually read-noise limited. Thus the binning results in improved detection limits while acceptable spatial resolution of the membrane is still maintained. For all data presented here, the CCD temperature is maintained at -110 OC, and the membrane exposure time (for signal integration) is varied from 15 min to 2 h. CCD detectors respond to cosmic rays and background radiation. As these events are very energetic, each event can produce thousands of electron^.^^^^^ These events limit the maximum practical observation time to under 4 h. The electrophoretic bands are several detector elements wide in both theXand Ydimensions and so can bedistinguished from intense cosmic ray induced "point spikes". A software routine written in the PMIS macro language removes the spikes and uses a median filter to improve the signal-to-noise ratio. All spikes 100 counts greater than the average of the eight neighboring detector elementsare removed and replaced with an average of the eight neighboring elements. The median filter, consisting of the averageof the eight neighboring pixels, is applied to the entire image. Both the raw data and the raw background data are treated by the same algorithm before background subtraction. Figure 3 shows raw and processed membrane images for a separation of tritiated amino acids, with both images having the same intensity scale. The images have been reversed so that the lighter areas indicate less light and darker areas indicate more light. Figure 3A is background-substracted only and shows a large number of singleand double-detector element spikesdenoted by the appearance of dark spots. The lack of spots in Figure 3B demonstrates the efficiency of the spike removal routine. Once the images have been processed as described above, the CCD rows containing the capillary trace are extracted and a standard electropherogramis reconstructed using Excel for Windows. For each electrophoretic row, three rows of Am!YtkalChemIsby, Vd. 66, No. 14, Ju& 15, 1994
2385
Cysteine-containingpeptides are labeled with [3sS]cysteine by forming a disulfide linkage. Benzofuroxan is used to form the disulfidebond.26 The reaction is completed in the running buffer. Peaks are identified by reacting the peptides individually with [Wlcysteineand comparingthe retention times of the individual labeled peptides to peak retention times of the peptide mixture. The peptides were purchased from Sigma Chemical Co. (St. Louis, MO) and American Peptide Co. (Sunnyvale, CA). All other chemicals are from Aldrich Chemical Co. (Milwaukee,.WI). Borate buffer is used for all separations; the peptide and ATP separations used a pH 8.4,61.5 mM (total borate) borate buffer, and the amino acid separation used a pH 10.0, 50 mM borate buffer. The pH is adjusted with sodium hydroxide.
Flgwe 3. (A, lop) Raw image of the membrane for a separation of five Mtiatedamlno adds. (B, Bottom) Processedimageof the membrane after spike removal and median filtering.
CCD data are exported into an Excel spreadsheet, summed into a single row, and plotted against time. All signal-tonoise calculationsare based on peak height. For the membrane images shown, images are exported as an 8-bit TIFF to Core1 Photo-Paint for reversal and printing. Reagents. Radiochemicals are from Amersham Co. (Arlington Heights, IL). The [3SS]methionine(CatalogNo. SJ.1515) is in an aqueous solution stabilized with 2-mercap toethanol and pyridine-3,4-dicarboxylic acid. The [3sS] cysteine (Catalog No. SJ.232) is in an aqueous solution containing potassium acetate and dithiothreitol. The nonstabilized cysteine is used to radioactively label peptides, as the stabilizers could interfere in the derivatization reaction. The tritium-labeled amino acid mixture (Catalog No. TRK.550) and a-32P-labeledATP (Catalog No. PB. 10384) are also from Amersham. All amounts of radionuclide are based on the manufacturer specifications adjusted for radioisotopic decay.
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2386 A m M i ~ l C b m i s m ,Vol. 66, No. 14, July 15, 1994
RESULTS AND DISCUSSION Two of the most important characteristics of this system are separation efficiency and LOD. Individual radionuclides are expected to have greatly varying characteristics and different optimum conditions, and so each radionuclide is discussed separately. The amounts of sample injected and LODs are reported in terms of mass as opposed to concentration as all membranes are dried after fraction collection before imaging and so the mass of the analyte is the important parameter. Sulfur-35. 3sSis one of the most important radionuclides because it is used in biological systems to label methionineand cysteine-containingpeptides and proteins. Figure4 shows the electropherogram of 66 amol (3.7 Bq) of 3sS-labeled methionine. The LOD, calculated using three injections, each based on the sum of three 1-h image exposures, yields a detection limit for 35Smethionine of 17 amol(0.94 Bq). This can be further improved using longer integration times or multiple exposures. The linearity of response of the membrane has been tested using a series of injections of 35S-labeledmethionine. The exposure time for all but the 66-am01 injection is 15 min. The membrane produced by the 66-am01 injection is exposed for 1 h and the results converted to a 15-min equivalent exposure (the background-corrected results are divided by 4). The system response is determined by summing the peak in the image and subtracting an equivalentlysized background signal from the membrane image between the traces (see Figure 2). The response versus labeled methionine is shown in Figure 5; the scintillator response is linear from 66 amol to 11 fmol(9 = 0.996). The response of the membrane for the highest mass injected, 33 fmol, has a significant deviation from the best-fit line. This injection represents injecting the straight methionine standard solution from Amersham; whether the deviation is caused by a matrix effect or membrane saturation has not been determined. Five replicate injectionsof the 0.67 fmol (37 Bq) standard gave a mean of 1560 counts with a RSD of 15%. This uncertainty is most likely caused by the manually timed gravity injections and irreproducibility in membrane coating. In order to demonstratea separation of labeled peptides, we separated a seriesof cysteine-containingpeptides and linked radiolabeled cysteine to the peptides with the formation of a ~
~
~
~~~~~~~
(26) Shipton. M.; Brocklehurst, K. Biuchem. J. 1977. 167, 79-10.
I
Methionine
250
100
50
0 -50
I
0.00
2.00
6.00
4.00
8.00
10.00
12.00
time (mln) Flgure 4. Reconstructed electropherogram of 66 amol of @Wabeled methionine. 5x10'
4x1' 0
efficiencyof the CE separationsfor both systems. The number of theoretical plates (-60 000) for [35S]methionineindicates that the separation efficiency is typical of on-line CE separations. For the cysteine-containingpeptides,the number of theoretical plates for the radiolabeled peptides ( 110 000 for C*-CDPGYIGSR-NH2) compared to on-line UV detection (-40 000) under these conditions indicates that the peptides bind to the membrane with little postcolumn broadening. Tritium. Tritium is one of the lowest energy j3- emitters used in biological investigations. Because of the ubiquitous nature of hydrogen, it can be used to label most compounds of biological interest. Its detection is much more difficult than the other radionuclides; for example, autoradiography of tritium in gels is problematic because the low-energy ,3particles do not penetrate the gel to expose the film. Liquid scintillationcounting is one of the most successfulapproaches to detect this radionuclide. We have separated and detected a mixture of five tritiumlabeled amino acids. The resulting electropherogramis shown in Figure 7. The lysine peak is not completely resolved and is broader than the other amino acid peaks. Both the membrane and the lysine carry a positive charge at the separation pH. This likely reduces the lysine binding interaction relative to that of the other amino acids. ACE run using UV detection at 185 nm does not show the lysine peak broadened relative to that of other amino acids. Because of the peak overlap and broadening, the peaks for lysine and proline are omitted from the calculation of average tritium detection limits and separation efficiency. For leucine, phenylalanine, and tyrosine, the average detection limit is 8 fmol of 3H for a 1-h exposure. Phosphorus-32. 3zPis commonly used to label nucleotides and monitor the phosphorylation of proteins. Unlike 35Sand 3H, there are previous reports of the detection of 32Pusing CE, allowing comparison of detection limits. An electropherogram of approximately 300 zmol of 32P-labeledATP is shown in Figure 8, yielding an 88-zmol (0.03 Bq) LOD for a single 2-h exposure. This represents nearly a 2 order of magnitude decrease in LODs compared to previous ~ o r k . ~ , ~
-
3x10 '
c 3 2x104
1V
0
2
4 fmol of
6
a
10
12
labeled methionine
Flgure5. Response of the system as a function of labeled methionine injected onto the capillary.
disulfide bond. Figure 6A,B shows the separation of four peptides with the electropherogram obtained using a single 1-hexposureof the membrane. The high efficiencyseparation demonstrates that peptides containingbetween 4 and 10amino acids can be separated and detected with this system. A total of 3 fmol of radiolabeled sulfur has been injected in the electropherogram shown in Figure 6, and so the integrated peak area of all peaks is from this 3 fmol of 35S. With the assumption of equal response for all 3SS-containing compounds,several peptides are present in the 100-200 amol range. The broad peak between 9 and 10 min is due to impurities in the [35S]cysteine. The radiochemical 'purity specification is 90% for this lot when purchased and degrades during storage due to interaction of the sample with the j3emission. Other unidentified peaks are thought to be products of disulfideformation between radiolabeled cysteineand other thiol-containing impurities and degradation products. We compareddata from UV detection to the radiochemical detection using similar capillary, buffer, and injection parameters to determine the effect of the membrane fraction collection on postcolumn band broadening. Table 2 lists the
AnaWcai Chemistry. Voi. 66, No. 14, JuEy 15, 1994
2807
ine
5500
4500
3500
c 3 2500 ,
cystine
so04 0.00
2.00
4.00
6.00
8.00
10.00
12.00
timo (min) 700 600
200 100
0 6.00
7.00
9.00
8.00
10.00
11.00
timo (min)
FlaUo 6. (A, Top). l%ctmPkwam of [ % ] W t W W pept#ea. (B, Bottom)Oetd of the e k Q t r o p h m x ” . A total of 3 hnol of Injected onto the CE column. Tabla 2.
theoretical plates nuclide
chemical form
32P
ATP
35s
Met peptides Phe Tvr
”S 3H 3H
detection limit (amol of nuclide) 0.09 17 17 8000 8000
radiochemical
UV
detection
detection
80800 60000 110000 70000 70000
nottested nottested 30000 30000 40000
In conclusion,we have demonstrated a novel radionuclide detection system that enables weak 6 emitters to be analyzed with excellentlimitsof detection andhigh separation efficiency. One of the limitations of the system is that the membrane binding properties only allow a select range of analytes to be examined. The Millipore ImmobilonPsq/CD (Experimental Section) works well for peptides and most amino acids, but not for positively charged amino acids. A wide range of protein binding membranes arecommercially available, and it is only in the area of analyzing small ions that potential problems arise due to the lack of a suitable membrane. The current LODs of the membrane fraction collection system represent large improvementscompared to on-line CE 2988
Analytical chemlsby, Vd. 66, No. 14, JUEL 15, 1994
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radiochemicald e t h n systems. Unlike the on-liqesystems, the light collection efficiency and membrane scintillator efficiency are far from their theoreticallimitsin the postcolumn system, and so additha1,improvements should be pwibk. As one exampie, a t a p e d image reduction fiber (optical minifier) is being examined to improve light collection efficiency approximately 10-fold. Increases in light collection should yield a lintar improvement in detection limit until the scintillationpraccssfrom a singleradionuclidedecay produces enough photons to bring the event over the CCD read-noise level, an approximately 100-fold improvement. The 15% RSD between replicate methionine injections is, at least in part, due to difficulties in reproducibly applying scintillatorto the membranes. We are currently investigating using a second capillary to deliver a known amount of radiolabeled standard at a preset distance from the separation capillary. The advantaw of the approach are severalfold. This will allow variations in scintillator efficiency to be normalized between membranes and will greatly facilitate automated routines to find and reconstruct the electrophero-
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Flgure 7. Electropherogram of a tritium-labeied amino acid standard solution. Each amino acid 1s present with equal acthrity. 140
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Flgure 8. Electropherogram of a 300-zmol injection of 32P-labeiedATP.
grams from the images. Lastly, the light collection efficiency from the camera lens pair is not uniform across the membrane; a smaller fraction of the emission from a point at the corner of a membrane is collected than from a point in the center. The main effect of this variation in collection efficiency is a "curved" baseline that "peaks" once per minute (corresponding to one pass across the membrane). This effect is visible in Figures 6 and 8. Although the use of analyte standards allows quantitative analysis to be performed even with this effect, a second capillary will allow the effect of nonuniform light collection efficiency across the membrane to be corrected. Even without these refinements, the current system affords flexibility in the trace-level assay of a variety of radionuclides. A significant advantage of this detection approach is that it is nondestructive. If the LODs reported are not low enough for a particular assay, the membrane can be repeatedly imaged, with the limitation being the stability of thescintillation process and the eventual decay of the radionuclide. This ability affords unprecedentedflexibilityin dynamicallyobtaining the required LODs after the CE separation has been performed.
ACKNOWLEDGMENT We thank Dr. Malcolm Pluskal of Millipore for providing the membranes for this research and for many useful discussions, Garret Forbes of the University of Illinois Chemistry Department for his help in examining a number of scintillator coatings, and Dr. Steven Pentoney, Jr. of Beckman Instruments for many helpful discussions. In addition, the donation by Millipore of the Quanta 4000 CE system is gratefully acknowledged. This work was supported by the National Institute of Mental Health (1R03MH49640), a National Science Foundation Young Investigator Award (CHE 92-57024), the David and Lucile Packard Foundation, and the Searle Scholars Program/Chicago CommunityTrust. Received for review November 22, 1993. Accepted April 13, 1994."
e Abstract
published in Aduance ACS Abstracts, June 15, 1994.
Analjltical Chemistty, Vol. 66,No. 14, July 15, 1994
2389
