Native, uncoloured, proteins can be focused in a column containing

a fluorescent packing material, using hydrodynamic flow and

a counteracting non-linear electric field, and imaged along the length

of the channel by fluorescence quenching

Ansell, Richard J.

Findlay, John B. C.

Ivory, Cornelius F.

Keen, Jeffrey N.

Myers, Peter

Tunon, Pilar Gonzalez

Wang, Yating

English

MURAL - Maynooth University Research Archive Library

COMMUNICATION www.rsc.org/analyst | AnalystElectrophoretic field gradient focusing with on-columndetection by fluorescence quenching†Richard J. Ansell,*a Pilar Gonzalez Tun˜on,ad Yating Wang,a Peter Myers,ae Cornelius F. Ivory,bJeffrey N. Keenc and John B. C. FindlaycReceived 4th September 2008, Accepted 10th December 2008First published as an Advance Article on the web 24th December 2008DOI: 10.1039/b815446eNative, uncoloured, proteins can be focused in a column containinga fluorescent packing material, using hydrodynamic flow anda counteracting non-linear electric field, and imaged along the lengthof the channel by fluorescence quenching.Electric field gradient focusing1–4 (FGF, also known as electric fieldfocusing or electromobility focusing) is an emerging technique for theseparation of ions based on their electrophoretic mobility ina column, where a hydrodynamic flow in one direction is opposed bya curved electric field. It is a member of the family of equilibriumgradient methods, also including isoelectric focusing (IEF), andtemperature gradient focusing (TGF),5,6 and like these has beenadapted to a microchannel/chip format.7–9 It offers an alternative toconventional electrophoretic techniques for protein separation andproteomics.Focusing techniques are potentially more sensitive than straight-forward electrophoretic techniques (native PAGE, CE) where thefield is constant and broadening increases with migration distance.FGF also has a potential advantage over conventional protein elec-trofocusing techniques. In gel IEF and capillary IEF a constantelectric field is opposed by a pH gradient, and focusing occurs at theisoelectric point of the protein, which often causes problems withprotein precipitation. In FGF the pH is constant throughout thechamber and focusing does not occur at the pI, so that higher sampleconcentrations may be accommodated.The FGF instrument designed by Lee et al. and Woolley et al. isbased on a shaped ionically conducting membrane surrounding theseparation channel.10–15 As the membrane becomes narrower, the fieldstrength increases. The design adopted by Ivory et al. uses an array ofelectrodes, separated from the separation channel by a dialysismembrane, and under individual control, which produces an electricfield which can be shaped at will16–18 (Fig. 1). Because of the capacityto change the electric field during a run, this version of the techniquewas dubbed dynamic field gradient focusing, DFGF. By dynamiccontrol of the shape of the electric field, the point at which an analyteion focuses may be moved up or down and analytes teased apart.aSchool of Chemistry, University of Leeds, Leeds, UK. E-mail: chmrja@leeds.ac.uk; Fax: +44 (0)113 3436565; Tel: +44 (0)113 3436415bDepartment of Chemical Engineering, Washington State University,Pullman, WA, USAcFaculty of Biological Sciences, University of Leeds, Leeds, UKdYork Bioanalytical Solutions Ltd., York, UKeDepartment of Chemistry, University of Liverpool, Liverpool, UK† Electronic supplementary information (ESI) available: experimentalconditions for synthesis of F254-containing cross-linked polyacrylamide.See DOI: 10.1039/b815446e226 | Analyst, 2009, 134, 226–229Other FGF designs have included focusing in a dialysis membranewith a conductivity gradient established along its length by a coun-terflowing low ionic strength buffer outside the membrane.19 Thisprinciple can be usefully adopted in Ivory’s instrument also.18In monitoring the separation on-column, Ivory and co-workershave mostly used coloured compounds, including coloured and dye-labelled proteins,16,17 and optical camera detection, while the Lee andWoolley groups have mostly used fluorescent/fluorescent-labelledproteins and laser-induced fluorescence.10–15 Of course this excludesthe vast majority of native proteins, and a similar limitation is foundin work up to now on microchannel IEF and TGF.5,6 Fluorescentlabelling of proteins complicates mass spectrometric analysis and canchange the electrophoretic mobilities of proteins (particularly if theFig. 1 Exploded cross-sectional schematic of the front block, spacer,and middle block, together with the voltage profile used in this work: (a)running buffer flow enters front via 1/1600 tubing and threaded frittedadaptor; (b) 5.7 cm long separation channel containing packing material;(c) dialysis membrane; (d) running buffer exits; (e) spacer with 6.7 cmlong cut channel; (f) 5.7 cm long electrode channel cut in back block; (g)21 platinum wire electrodes (stretched between electrode pins); (h)coolant flow enters via barbed adaptor; (i) coolant exits; (j) glass slide forintroducing UV.This journal is ª The Royal Society of Chemistry 2009Fig. 3 Rear chamber (a) and front chamber (b) showing the device inplace and the tubing for fluidics. The electrode connections are not inplace for clarity.label is ionisable), so that in the case of inhomogeneous labellingpeaks would be artificially broadened. Monitoring native proteins byUV absorbance, however, would require UV-transparent materials,e.g. quartz glass, which add significantly to the cost of prototypedevices, while the array of electrodes required in DFGF would seemto preclude UV transmittance perpendicular to the separationchannel along the whole of its length.Both the Lee and Woolley groups and Ivory et al. have also studiedthe conditions under which analytes are eluted from the separationchannel,13,17 using post-column detectors, which would be necessary,for example, to enable structural identification of unknown proteinsby mass spectrometry. An ideal system might combine on-channeland post-channel detection, in order to ensure that analytes are teasedapart and eluted one at a time.A DFGF system allowing on-column detection of native, uncol-oured proteins is presented here for the first time, based on quenchingof fluorescence of manganese-activated zinc silicate (F254 indicator,EM Science, lex¼ 254 nm, lem¼ 500 nm, a material commonly usedin fluorescent thin-layer chromatography plates). The mechanism ofquenching, as in TLC, is the absorbance of the excitation radiation bythe analyte.20 The device was based closely on the DFGF system usedby Tun˜on et al.18 (Fig. 1). The rear block, containing the electrodearray, also contained a 50  50  1 mm UV glass slide (Comar)which extended 10 mm into the Plexiglass (poly(methyl methacry-late)) and protruded 40 mm out of the back. The edge inside the blockwas exposed to the coolant channel, and the 0.25 mm diameterplatinum wire (Aldrich) electrodes were stretched at intervals of 0.100between channels either side of the glass, attaching to the pins of twomale–female connector strips (Samtec). There was a 0.5 mm gapbetween the edge of the glass slide and the wire electrodes, and a 0.25mm gap between the wires and the surface of the rear block. With the3.2 mm thick black Teflon (polytetrafluoroethylene) spacer in place,this meant a gap of 3.45 mm between the electrodes and the dialysismembrane (Spectrum, Spectra/Por cellulose acetate membrane,molecular weight cut-off ¼ 100 Da) which separated the coolantchannel from the separation channel.The 57 mm long, 1 mm wide and 0.5 mm deep separation channelmachined into the front block was packed with cross-linked poly-acrylamide beads containing 0.5% w/w F254 indicator (Fig. 2). Thebeads were prepared by suspension polymerisation using an aqueousreacting phase and organic dispersing phase in the presence of theF254 indicator particles (see ESI†).21 Although the particles are not allspherical and there is a distribution of particle sizes, this is notexpected to influence the efficiency of the focusing. Unlike inFig. 2 Polyacrylamide packing material containing F254 indicator.This journal is ª The Royal Society of Chemistry 2009chromatography, the packing in FGF is present only in order toreduce the diffusion of analyte in the separation channel. The pres-ence of differing paths through the channel is not detrimental sincethere will still nonetheless be only one point at which the hydrody-namic flow balances the electrophoretic force. Moreover, since theflow rate used here is very low (20 mL h1) the additional back-pressure generated by using a packing with a heterogeneous sizedistribution is also insignificant. The remaining dimensions of thedevice and details of connections and auxiliary pumps etc. wereexactly as in the previous work.18 The device was placed in a purpose-built cabinet with a rear compartment and front compartmentseparated by a black-painted Plexiglass screen containing a gap intowhich the device fitted (Fig. 3). The rear block (with the glass slideprotruding) was in the rear compartment close by a 6 W 254 nm lamp(UV Products, UVS 16-EL) and the front block, containing theseparation channel, was in the front compartment where it was facedat a distance of 28 cm by a CCD camera (Diagnostic InstrumentsSPOT Insight 4, with a Nikon 50 mm 1 : 1.4 lens). Photographs werecollected using the SPOT Advanced software running on a Pentium 4PC, converted to .jpg format and intensity histograms were derivedusing Scion Image processing software (Scion Corporation).By trial and error the optimal position of the UV lamp was foundto be 4 cm behind the rear edge of the glass slide, and 2 cm to the sideof it, such that UV light was incident on the slide at a grazing angle ofapproximately 20%.Proteins were introduced into the separation channel via aninjection valve (Upchurch Scientific) with a 10 mL sample loop,introduced into the pH 8.6 100 mM Tris run buffer at a flow rate of20 mL h1. The coolant buffer was pH 8.6 1 mM Tris, pumped in thecounter direction at5 mL s1. The electric field shown in Fig. 1 wasswitched on once the protein had entered the channel. Fig. 4a and 4bshow the focusing of horse liver alcohol dehydrogenase (HLADH,from which NAD had been removed by gel filtration) and horseshoecrab hemocyanin (HC) injected separately, after 5 h focusing. Thesignal plots appear quite noisy for several reasons: the electrode wiresacross the coolant channel block out some of the excitation lightcausing dips in the emission intensity at regular intervals; the emission(before quenching) is more intense in the centre of the channel than atAnalyst, 2009, 134, 226–229 | 227Fig. 4 Focusing of (a) HLADH (100 mg) and (b) hemocyanin (100 mg),using the FGF21 system, 20 mL h1. Right-hand side corresponds to theinlet (top) of the device. Images are from colour photographs convertedto greyscale with equal weighting of RGB channels. Intensity profiles arecalculated from the greyscale images using Scion Image. Image (c) showsthe column prior to injection and (d) after focusing a mixture of bothproteins (25 mg each). Image (e) is calculated by the software by sub-tracting 0.57  (c) from (d) and optimising the contrast.the extremities (Fig. 4c), possibly because the glass waveguide chan-nels are in light which is more intense at this point or because of‘shadows’ cast by the ends of the channel; and the emission may alsovary due to inhomogeneities in the packing along the channel.Nonetheless, peaks due to the focused analyte can be seen clearly ineach case.The major (EE) fraction of commercial HLADH (Sigma A9589)has a pI of 8.1 and Mr ¼ 80 000.22 Commercial HC (horseshoe crabtype VIII, Sigma H1757) is a mixture of isoforms with pIz 6 andMr¼ 72 000.23,24 Therefore HC is expected to have higher mobility at pH8.6; the mobilities as measured by CE in 20 mM pH 8.6 Tris are 0.14 104 cm2 V1 s1 for HLADH and 2.05 104 cm2 V1 s1 for HC(ESI†). HC indeed focuses further up the channel (1.9 cm from theinlet vs. 2.3 cm). HLADH, having a lower mobility, proceeds furtherdown the channel until it encounters a sufficiently steep electric fieldthat the product E  mep balances exactly the hydrodynamic flow.228 | Analyst, 2009, 134, 226–229The peaks in both cases are quite broad, and when a 50 : 50 mix wasinjected it was not resolved. However, on further dilution tworesolved peaks were apparent (Fig. 4d). Although the width offocused bands should theoretically be independent of concentra-tion,16 it has been observed previously that the width increases withanalyte amount, which may be attributed to the concentrated analyteions distorting the local electric field.25 The separation could be bettervisualised by subtracting the background photograph taken pre-injection as shown in Fig. 4e.This proof-of-principle study has shown that native, uncolouredproteins can be visualised on-column during focusing by FGF/DFGF. The visualisation technique should be applicable to allproteins as they all absorb at 254 nm. FGF is applicable to allproteins as long as the electrolyte pH differs from their pI – acididcproteins can be separated in normal polarity FGF as described hereand basic proteins with reversed polarity. The two proteins here havesignificantly different electrophoretic mobilities; however, DFGF hasbeen shown to be capable of resolving even the two oxidation statesof myoglobin, whose mobilities are much closer under the conditionsused. 25 mg amounts of the proteins were readily detected and Fig. 4esuggests that the detection limit could be an order of magnitudelower, dependent on the extinction coefficient of the particularprotein. With optimisation of the excitation intensity, columndimensions and detection parameters it should be possible to improvethis further. The resolution of the particular proteins studied was notas good as expected based on studies of coloured proteins in earlierwork,18 which may be due to interactions between the proteins andthe fluorescent packing material. Similar broadening of the focusedbands is observed when silica is used as the column packing. Futurework will investigate different packing materials containing the F254indicator and different proteins which possess greater homogeneity.This work was funded by the BBSRC, grant number BB/C006771/1.Notes and references1 C. F. Ivory, Sep. Sci. Technol., 2000, 35, 1777–1793.2 R. T. Kelly and A. T. Woolley, J. Sep. Science, 2005, 28, 1985–1993.3 W. S. Koegler and C. F. Ivory, Biotechnol. Prog., 1996, 12, 822–836.4 J. G. Shackman and D. Ross, Electrophoresis, 2007, 28, 556–571.5 J. G. Shackman, M. S. Munson and D. Ross, Anal. Bioanal. Chem.,2007, 387, 155–158.6 G. J. Sommer, S. M. Kim, R. J. Littrell and E. F. Hasselbrink, Lab ona Chip, 2007, 7, 898–907.7 D. N. Petsev, G. P. Lopez, C. F. Ivory and S. S. Sibbett, Lab ona Chip, 2005, 5, 587–597.8 P. Myers and K. D. Bartle, J. Chromatogr., A, 2004, 1044, 253–258.9 L. K. Ista, G. P. Lopez, C. F. Ivory, M. J. Ortiz, T. A. Schifani,C. D. Schwappach and S. S. Sibbett, Lab on a Chip, 2003, 2003,266–272.10 P. H. Humble, R. T. Kelly, A. T. Woolley, H. D. Tolley andM. L. Lee, Anal. Chem., 2004, 76, 5641–5648.11 K. F. Warnick, S. J. Francom, P. H. Humble, R. T. Kelly,A. T. Woolley, M. L. Lee and H. D. Tolley, Electrophoresis, 2005,26, 405–414.12 P. H. Humble, J. N. Harb, H. D. Tolley, A. T. Woolley, P. B. Farnsworthand M. L. Lee, J. Chromatogr., A, 2007, 1160, 311–319.13 R. T. Kelly, Y. Li and A. T. Woolley, Anal. Chem., 2006, 78, 2565–2570.14 J. K. Liu, X. F. Sun, P. B. Farnsworth and M. L. Lee, Anal. Chem.,2006, 78, 4654–4662.15 X. F. Sun, P. B. Farnsworth, A. T. Woolley, H. D. Tolley,K. F. Warnick and M. L. Lee, Anal. Chem., 2008, 80, 451–460.16 Z. Huang and C. F. Ivory, Anal. Chem., 1999, 71, 1628–1632.17 J. M. Burke and C. F. Ivory, Electrophoresis, 2008, 29, 1013–1025.This journal is ª The Royal Society of Chemistry 200918 P. G. Tun˜on, Y. Wang, P. Myers, K. D. Bartle, L. Bowhill,C. F. Ivory and R. Ansell, Electrophoresis, 2008, 29, 457–465.19 Q. G. Wang, S. L. Lin, K. F. Warnick, H. D. Tolley and M. L. Lee,J. Chromatogr., A, 2003, 985, 455–462.20 J. W. Sease, J. Am. Chem. Soc., 1947, 69, 2242–2244.21 P.-O. Larsson and K. Mosbach, Methods in Enzymology, 1976, 44,183–190.This journal is ª The Royal Society of Chemistry 200922 I. Hubatsch, P. Maurer, D. Engel and H. W. Adolph, J. Chromatogr.,A, 1995, 711, 105–112.23 M. Brenowitz, C. Bonaventura, J. Bonaventura and E. Gianazza,Arch. Biochem. Biophys., 1981, 210, 748–761.24 J. Maddaluno and K. F. Faull, Biochem. Biophys. Res. Commun.,1999, 264, 883–890.25 N. I. Tracy and C. F. Ivory, Electrophoresis, 2008, 29, 2820–2827.Analyst, 2009, 134, 226–229 | 229

Electrophoretic field gradient focusing with on-column

detection by fluorescence quenching

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Electrophoretic field gradient focusing with on-column detection by fluorescence quenching

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