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Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides
J. Biochem. Biophys. Methods 39 (1999) 93–110
Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides Qinglu Mao, Janusz Pawliszyn* Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2 L 3 G1 Received 26 November 1998; received in revised form 7 January 1999; accepted 7 January 1999
Abstract Whole column imaging detection has been developed for capillary isoelectric focusing (CIEF) of proteins and peptides. In this imaged CIEF technique, a solution of sample and ampholytes was introduced into a short (4–5 cm), internally coated capillary stabilized by a cartridge. After applying high DC voltage, the isoelectric focusing process takes place and the focused zones are monitored in a real-time mode using the imaging detectors developed. Three types of imaging detectors have been developed including refractive index gradient, laser-induced fluorescence (LIF), and absorption. Of these, absorption imaging detection is the most practical at the present time due to its quantitative ability and universal characteristics. Whole column imaging detection eliminates the mobilization step required for single point detection after the focusing process. Therefore, it provides a fast analysis speed (3–5 min for each sample), and avoids the disadvantages associated with the mobilization process, such as distortion of pH gradient and loss in resolution. In this paper, we review the methodology of imaged CIEF as well as progress in instrumental development, IEF performed on a microchip, and the application to protein and peptide analysis. 1999 Elsevier Science B.V. All rights reserved. Keywords: Capillary isoelectric focusing; Whole column imaging detection; Protein and peptide analysis
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1. Introduction Isoelectric focusing (IEF) is a well known high-resolution technique for biochemical separation [1,2]. It is routinely used for characterization of biological extracts, monitoring protein purification, evaluating the stability or microheterogeneity of protein therapeutics, and determination of protein isoelectric points. Capillary isoelectric focusing (CIEF) [3–7], which was first reported by Hjerten and Zhu [3], combines the high resolving power of conventional gel IEF with the automation and quantitation advantages of capillary electrophoresis (CE) instrumentation. It offers several advantages over traditional gel-based techniques, including ease of automation, quantitation, speed, and detection by UV absorbance (eliminating laborious staining and destaining). In the past decade, CIEF has usually been carried out in commercial CE instruments which have a 20–60-cm long capillary and an on-column UV absorbance detector. Samples, usually proteins, are mixed with carrier ampholytes and introduced into the capillary between the catholyte and anolyte. A high DC voltage is then applied and, as a result, a relatively stable pH gradient is established along the capillary axis by the carrier ampholytes [8]. For a ‘two-step’ mode [4], proteins are first focused at the position where their net charge is zero, i.e. where their isoelectric points (pI values) are the same as the pH values. After focusing, all the focused protein zones are moved through the detection point. Methods using chemical, electrophoretic and hydrodynamic mobilization are well developed. Alternatively, mobilization can be performed during the focusing process with what is called ‘one-step’ CIEF. Proteins are focused and driven towards the detection point by electroosmotic flow (EOF) [9,10]. ‘One-step’ CIEF provides short analysis time, however, it gives lower resolution than mobilization by salt. The use of CIEF for both research and industrial (e.g. quality control, clinical diagnosis) analysis of proteins and peptides is now well established, with reported R.S.D. values for precision in the 2–3% range. The requirement for mobilization to facilitate single-point detection is unique to IEF performed in capillary format. Problems associated with the mobilization process may be encountered, including long analysis time, high risk of protein precipitation, distortion of pH gradient and uneven resolution due to a non-uniform mobilization speed. For example, although the run time of CIEF for a single sample is faster than that of slab gel IEF, CIEF throughput is lower than that with the latter since CIEF is run in a single capillary while slab gel IEF can simultaneously run several samples in different lanes. Also, the conditions and the speed of the mobilization process have to be optimized for different samples to achieve the highest resolution [11]. For these reasons, it is preferable to implement whole capillary imaging detection for CIEF so that the focusing process takes place, as in the slab gel IEF, free from any disturbance, such as the influence of electroosmotic flow (EOF) or hydrodynamic flow. To overcome the drawbacks of single point detection CIEF caused by the mobilization, much effort has been focused on the development of whole column detection for CIEF that eliminates the requirement of mobilization. Whole column detection can be realized by pulling the separation capillary through the detection window [12], however, several limitations are obvious. An additional moving device is required, dynamic noise is relatively high, and distortion of zones still exists due to movement of capillary.
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Another example is a spatial-scanning laser fluorescence detection for CIEF reported by Beale and Sudmeier [13]. This method enables monitoring of the dynamic focusing process, but the slow scan speed (over 15 s for one scan of 8 cm capillary) limits its detection for a fast focusing process (e.g. about 3 min under 600 V/ cm). In the past few years, imaging detection has been found to be ideal for whole column CIEF detection [14]. Three types of imaging detectors have been developed: refractive index gradient (concentration gradient) [15–18], fluorescence [19] and absorption [17,20]. Imaging detection, which provides a real-time monitoring has proven useful in biochemical separation techniques, such as electrophoresis. Two types of imaging detection are used: (1) photograph the focused bands which have luminescent light emission. One well-known example is DNA gel electrophoresis incorporating ethidium bromide [21]. (2) Record transmitted light passing through or fluorescent emission from focused bands. This second type of spatial imaging detection has been developed for capillary electrophoresis. Sweedler and Zare [22] constructed an LIF detector using a charge-coupled device (CCD) camera in a time-delayed integration mode to image the last few centimetres of a separation capillary. Whole column imaging detection for CIEF, known as imaged CIEF, has benefited CIEF analysis significantly. Advances in such imaged CIEF techniques and the performance improvement obtained from the commercial instrument (iCE280, Covergent Bioscience, Etobioke, ON, Canada) have increased its potential application in biotechnology. This review will present a general description on both methodological and application aspects of imaged CIEF technique.
2. Principles of imaged CIEF
2.1. Description With CIEF, IEF is performed in free solution in a capillary format, with the absence of flow. All focused zones are detected by a real-time imaging detector. Unlike that used in slab-gel IEF, where the focused bands are silver-stained and photographed, the principle of imaged CIEF is based on the physical-chemical properties of the focused zones, such as absorption, fluorescence and refractive index gradient (concentration gradient) etc. In single point detection, a detection window is created. In imaged CIEF, an optical system is constructed for whole column detection as shown in Fig. 1. Light from a light source is expanded and projected onto the whole separation capillary, and either transmitted or fluorescence light is detected by a linear or 2-D area sensor, such as CCD and photodiode array (PDA). To simplify the optical alignment, a short separation capillary (5 cm) is favoured. The bare capillary is internally coated in order to minimize electroosmotic flow (EOF) and reduce adsorption of proteins to the column wall. A capillary cartridge is assembled to support the capillary, and is aligned with the imaging detectors. Two pieces of hollow fiber membrane are connected to the capillary ends to isolate the sample solution and the electrolytes, and to allow convenient sample introduction and stable focusing [23]. In imaged CIEF, the pI values of the proteins can be determined directly by their positions in the capillary due to a linear gradient inside
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Fig. 1. Instrument set-up of whole column imaging detection for CIEF. Absorption or refractive index gradient mode with camera (I) placed in the direction of illuminated light; fluorescence mode with camera (II) placed vertically to the direction of illuminated light.
the whole column [24]. A detailed description of the instrument set-up will be presented in Section 3.2 and Section 3.3.
2.2. Resolving power It is a fear that the short capillary may cause a decrease in resolution for imaged CIEF. For this reason, theoretical considerations on the feasibility of a short capillary are addressed here. For a sample zone focused in a capillary by the isoelectric focusing process, concentration has a Gaussian distribution with a variance s [1,2]: C 5 C0 exp(2pEx 2 ) / 2D
(1)
where C5C0 is the maximum concentration, p the mobility slope (2du / dx), E the field strength, D the diffusion coefficient and x position along the capillary.
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]]] D dx s 5 6 ] ]] E (2du)
œ
97
(2)
Using the criterion of three times the variance s for resolved adjacent proteins, the resolving power, DpI, of IEF in terms of s can be expressed as [1]: ]]]]] D d(pH) d(pH) DpI 5 3 ] ]] ]] E (2du) dx
œ
(3)
Eq. (3) shows that good resolution is favoured by a high field strength, a low diffusion coefficient, a high mobility slope dm / d(pH), and a narrow pH gradient. Of the variables, the diffusion coefficient and the mobility slope are intrinsic properties of the analytes, so only pH gradient and the field strength can be varied experimentally. In conventional CIEF, where long capillaries (12–60 cm) are used, pH gradients are more shallow than those seen in imaged CIEF with short capillaries. Satisfactory resolution, however, still can be obtained by adopting a higher field strength. Narrow fused silica capillaries have excellent heat dissipation and so allow high filed strengths (500–800 V/ cm) to be applied. When a narrow pH gradient of 6–8 and a high voltage of 3 kV is used, a resolution of about 0.03 pH units can be achieved with imaged CIEF performed in a 5-cm-long capillary [25]. This is a bit lower than that in optimized conventional single-point detection CIEF with a resolution of 0.01–0.02 pH units, however, the resolution is good enough for clinical analysis. Due to the short capillary, imaged CIEF attains equilibrium faster, e.g. within a few minutes (2–3 min).
3. Method and instrumental development
3.1. Refractive index gradient imaging detector Capillary isoelectric focusing achieves both concentration and focusing of sample components, and so a high concentration gradient is created at each boundary of separated zones inside the capillary. As a result, a refractive index gradient is present at each boundary. The focused zones can therefore be detected through refractive index gradient (concentration gradient) imaging detection. A typical instrument set-up can be constructed on the basis of either the Schileren shadowgraph method [16] or a dark field Toepler–Schliren system [17]. Generally, a well-collimated laser beam is focused onto the capillary column. Theoretically, the laser beam can be regarded as a bundle of infinitesimal light filaments which are parallel. Upon encountering a sample zone inside the capillary, the light filaments are refracted by the refractive index gradient present at each zone boundary, and bent out of their original directions. In the constructed systems, a He–Ne laser (at 633 nm wavelength) [17] or a visible diode laser (at 670 nm) [18] was used as the light source. Compared with the Schileren shadowgraph method, a dark-field Toepler–Schliren system is a more reliable system [17]. The latter is shown in Fig. 2. Its signal is positive and low noise is observed. The light intensity at the maximum deflection angle reaching
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Fig. 2. Instrument set-up of refractive index gradient imaging detector for CIEF based on a dark field Toepler–Schliren system. L1, L2 and L3 are three focusing lenses. Reprinted from Ref. [14] with permission.
the detector can be calculated from the sample concentration, C0 , the capillary length, l, and the zone width, 4s:
S
I0 p B L dn C0 lf I 5 ]] 0.48] ] ]] 2a n dC s 2 8l f
D
(4)
where n is the refractive index of the solution inside the capillary, L the inner diameter of the capillary, and C the protein sample concentration. The signal intensity of the detector is proportional to the refractive index gradient instead of refractive index itself. Refractive index gradient imaging detector is a real-time and universal process. A detection limit of 10 26 M was reached for all proteins tested under optimal conditions.
3.2. Laser-induced fluorescence imaging detector LIF detection is known as one of the most sensitive methods for CE detection [26]. In CIEF, focused protein zones labelled with fluorescent reagent can be detected using a fluorescence imaging detector [19]. As shown in Fig. 1, when the CCD camera is placed in the vertical position to the illumination light direction, fluorescence imaging is
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realized for CIEF. Basically, the laser beam from an argon ion laser was first expanded by either an optical fiber bundle or a microscopic objective lens, and then was focused onto the whole separation column. The fluorescence emission from the focused zones was detected by a CCD camera. A band-pass filter is placed in front of the camera lens to cut off scattering and background light. The wavelength and the band-pass width of the filter depend on the emission spectra of protein samples. The feasibility and performance of the imaged LIF-CIEF system was demonstrated with the proteins b-phycoerythrin, a-D-galactosylated FITC-albumin, insulin-FITC, and casein-FITC. The method is capable of separating and detecting 10 211 M protein samples. Similar LIF imaging detection systems can be found in other examples [13,21,27,28].
3.3. Absorption imaging detector A typical UV-VIS absorption imaging detection system is shown in Fig. 1 where the imaging sensor, camera (I), is fixed linearly to the illumination light direction. A xenon lamp, deuterium lamp, or tungsten lamp can be used as the light source, and a small grating monochromator covering 190–700 nm or a 280 nm bandpass filter can be used to obtain monochromatic light. A fused silica optical fiber bundle guides the light to project onto the CIEF separation column. Between the optical fiber bundle and the capillary column, a cylindrical focusing lens is used. The detector is a camera installed with a linear CCD or PDA. With a 2-D area sensor and a grating, absorption spectra for each focused zone can be obtained [20]. For UV-280 absorption imaging, the typical maximum absorption of proteins, a UV lens is used and the CCD sensor should be UV-sensitive. A capillary cartridge with a 5-cm-long capillary was used to perform CIEF. The absorption imaging mode clearly displays the concentration profiles of the individual focused zones. It is quantitative and universal for most proteins. If the optical alignment for imaged CIEF is optimized, peak broadening will not appear for the imaging of the focused zones after focusing is complete. Absorption imaging detection has become the most practical mode and been well developed. As a result, the commercial instrument is available now. In the following paragraphs, some basic points associated with the absorption imaging detector are presented.
4. Capillary cartridge and imaged CIEF procedures
4.1. Capillary cartridge In the early development stages of imaged CIEF, the cartridge used was very simple: one short capillary (4–5 cm) was connected with two electrolyte reservoirs at the two ends. However, this cartridge is not convenient for sample injection. A new cartridge, as shown in Fig. 3, has been developed for absorption imaging detection [25], and is also suitable for refractive index gradient detection. The separation capillary is internally coated, and its outer polyimide coating is removed. Two pieces of hollow fiber dialysis tubing are glued to the ends of the separation column. Two connection capillaries for sample introduction are glued to the other ends of hollow fiber membranes. Two glass
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Fig. 3. Schematic diagram of the capillary cartridge used for the commercial instrument for absorption imaged CIEF. Electrolyte tanks are affixed to one piece of glass, in which a 5033 mm slash was drilled for UV light to reach the separation column.
plates are used to hold the capillary and one piece of slit is used to cut off the stray light. The membranes that are immersed in the electrolyte reservoirs will allow the small ions like H 1 and OH 2 to pass freely and thus allow CIEF to perform normally. Using such a configuration, the introduction of the sample solutions mixed with carrier ampholytes can proceed without disturbing the electrolytes. In addition, for increasing the throughput, multiple capillaries can be used. A better future option will be the chip cartridge as described later.
4.2. Capillary coating The internal coating of capillary is very important in imaged CIEF. Due to its surface dielectric layers, fused silica capillary usually has a large EOF while a high voltage is applied. If EOF in a capillary is significant, the advantages of imaged CIEF will be lost. To ensure the focused zone remains static after focusing, the inner walls of capillary must be coated. Both static and dynamic coating have proven effective. Several polymer
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coatings provide pH stability and are suitable for CIEF. Examples are polyacrylamide, polyvinylpyrrolidone, polyethylene glycol, poly(vinyl) alcohol and fluorocarbon etc. [6]. Presently, two kinds of coated capillaries are adopted for imaged CIEF [29]. One is chemically coated with linear polyacrylamide [30] and another is physically coated with fluorocarbon. The commercially coated capillary with linear polyacrylamides shows over 100 reproducible runs for imaged CIEF. Fluorocarbon is more robust due to the physical coating and can be used for a long time. To improve performance, conditioning of the capillary is conducted before focusing. Usually, 0.1% methylcellulose solution is forced into the separation column by syringe pump for 20 min. Also, the sample solution is prepared containing 1% methylcellulose to increase the viscosity. With such a treatment, EOF is substantially reduced, and hundreds of runs are observed to be reproducible for the fluorocarbon-coated capillary [29].
4.3. Procedures for imaged CIEF To perform imaged CIEF, the electrolyte reservoirs are first filled with anolyte (H 3 PO 4 solution) and catholyte (NaOH solution), respectively. Then, sample solution is injected into the column cartridge. The commercial imaged CIEF instrument employs an eight port-two position HPLC injection valve for sample injection to the separation column [29]. It has two positions, one is loaded and another is injection. The injection port of the valve is linked to either a needle port for manual sample injection using a micro syringe, or a HPLC autosampler. The sample loop volume is 2.5 ml. The syringe pump is used to deliver sample and washing solution, and is kept running during the entire experiment. The sample injection and the imaged CIEF instrument are fully controlled by a PC computer. After the column is filled with the sample solution, a high voltage (1.5 or 3 kV) is applied to start the isoelectric focusing. The entire process usually takes less than 5 min in a 5-cm-long separation column. A reference image is taken after the sample is filled and stabilised. During the focusing process, images are consecutively taken to monitor the focused zones. Absorption electropherograms are obtained by automatically comparing to the reference image. The final pherogram gives a complete record of the focused zones without mobilization. After one run is finished, the column is washed by the wash solution (0.1% methylcellulose) and the system is ready for the next sample injection. In some cases, protein samples may have a high salt concentration, which compresses the pH gradient and has an impact on the separation pattern. In such case, desalting is necessary to perform using an on-line sample desalting device [23,25].
5. Performance of imaged CIEF
5.1. Reproducibility The reproducibility of imaged CIEF is defined by two parameters: (1) the position inside the column corresponding to pI value; (2) the peak height or peak area for quantitation. Theoretically, the focused zones should be reproducible due to the
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elimination of the mobilization. However, several factors affect the reproducibility, such as electroosmotic flow, hydrodynamic flow and the adsorption of sample to the capillary wall [30]. Electroosmotic flow may become higher after hundreds of runs for a given column since the coating on the inner wall of the column is subject to wear. The electroosmotic flow is usually toward the cathodic end of the column. Also, the solution within the open capillary can be driven by hydrodynamic flow. These factors must therefore be controlled to achieve maximal reproducibility. Fig. 4 shows three consecutive runs for one sample containing five pI markers. For five runs in the experiment, standard deviation of pI marker 8.6 is 0.05 pH units for the pH 2–11 carrier ampholytes, which corresponds to an R.S.D. of 0.6%. Normally, pI markers are added to protein samples as internal standards and the distances between the pI marker and the protein peaks are measured as the protein peak positions (relative positions), instead of the absolute positions along the column. By using the relative position, the standard deviations are less than 0.01 pH units providing R.S.D. of about 0.1%.
5.2. Detection limit CIEF usually concentrates proteins over 100-fold relative to that of the injected sample. Therefore, the detectability for protein should theoretically be higher than that of CE without preconcentration. However, the inability to detect at low UV wavelengths, due to carrier ampholyte absorption, limits the detectability of CIEF with single-point UV detection for analysis of proteins present at concentrations of 5–10 mg / ml or greater [5]. As for absorption imaged CIEF, the detection limit is a bit higher because of the use of CCD sensor. It provides a detection limit of 10 mg / ml for hemoglobin A1 and has a
Fig. 4. Electropherograms for three consecutive runs of five pI markers separated by absorption imaged CIEF. The focusing time was 3.5 min. pH 2–11 ampholytes were used. 0.5% TEMED was added to the sample.
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Fig. 5. Electropherogram of hemo control AFSC (partly glycolyted due to long time storage, indicated by small peaks in the left of four relatively high peaks) separated by imaged CIEF. pH 3–10 ampholytes were used. Left: anode, right: cathode.
baseline noise level of 2310 23 AU when 64 scans of a 2048-pixel linear CCD sensor are averaged as shown in Fig. 5. At high light levels, the shot noise is dominant [31] and mainly limits the detectability. In the commercial version of imaged CIEF, a Xe lamp and a linear CCD are used as light source and sensor, respectively. Due to its low well capacity, CCD is not ideal for absorption imaging detection as it has a very low read noise. Absorption imaging detection is best performed with an array sensor with the ability to discriminate differences of light intensity as small as possible. A specially designed PDA detector usually has a high well capacity of 80–90 M e 2 and makes both read noise and shot noise insignificant. As an example, PDA is recently reported to achieve a low detection limit for absorption imaging detection in CZE, comparable to or exceeding that of a commercial single-point detector [32]. By employing a PDA array and more stable light source, such as a D 2 lamp, instead of the Xe lamp, a lower detection limit would be achieved, thus making absorption imaging detection more comparable to the single-point detection.
6. CIEF performed on chip with imaging detection The microfabrication of analytical instrumentation has received great interest recently, offering a compact, reliable and inexpensive method for chemical and biological separations [33]. CIEF performed on microchip is of interest and is expected to have
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some great advantages over the conventional CIEF techniques in separation speed, compact size, integration and throughput etc. The feasibility of isoelectric focusing (IEF) performed on chip was demonstrated for the first time via absorption imaging detection in our group (Mao and Pawliszyn, submitted). Absorption imaging detection is ideal for chip IEF due to its universal and spatial detection mode. In most CE systems, laser-induced fluorescence is widely used, although limited by the need for labelling of proteins lacking fluorescence. Direct single point UV absorbance detection also is not suitable for chip IEF due to the requirement for mobilization. Instead of using the capillary cartridge, a chip cartridge for IEF was fixed in the optical path. Chip channels for separation were fabricated on a piece of quartz microchip using photolithography and chemically etching process (Albert Microelectronic Corporation, Edmonton, Alberta, Canada). The separation channel is 40 mm long, 100 mm wide and 10 mm deep, and was coated with linear polyacrylamide to reduce electroosmotic flow and decrease the adsorption of proteins to channel walls. Low molecular mass pI markers, substituted monomethylphenols, and the protein pI marker, myoglobin, were selected as model samples. Good performance of IEF has been achieved in microchip channels as shown in Fig. 6. The focusing process is reproducible and the resolution is similar to that of imaged CIEF in capillary format. Chip cartridge IEF has some advantages over the capillary cartridge one. It offers a smooth baseline with less spike noise and eases the optical alignment between the channel and slit due to the square geometry of the separation channel. Also, multiple channels can be more easily constructed to increase throughput than in a capillary cartridge [20,34].
Fig. 6. Separation of pI markers 7.4 and 6.6 on coated channel with chip cartridge. Concentration of each component is 4 mg / ml; pH 3–10 ampholytes were used and a high voltage of 3 kV was applied. Left: cathode, right: anode.
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7. Applications
7.1. Analysis of proteins and peptides The direct application of imaged CIEF is to analyze proteins and peptides. Many protein standard samples have been analyzed using absorption imaging detection. Examples are myoglobin, hemoglobin, cytochrome c, anti-a-acid glycoprotein, IgG, a-acid glycoprotein and monoclonal anti-a-antitrypsin transferrin [29,35,36]. Satisfactory separations have also been achieved for a number of real samples (unpublished data). Peptides can also be analyzed by imaged CIEF [29]. At 280 nm, only tyrosine and tryptophan have absorption, and sensitivity of UV absorption detector is low for many peptides. Fig. 7 shows an electropherogram of a peptide which contains only one tyrosine and no tryptophan. For this reason, a high concentration (2 mg / ml) was adopted for the analysis. However, due to the peptide being concentrated in the focusing, the peptide easily precipitates in less than 2 min after the focusing starts. By adding glycerol to the sample, its solubility can be enhanced and the focusing becomes possible, as shown in Fig. 7. Reproducible results can be achieved when 20% glycerol is added to the sample.
Fig. 7. Separation of peptides (Ac-Asp-Asn-pTyr-Ile-Ile-Pro-Leu-Pro-Asp-Pro-Gly-OH) by absorption imaged CIEF under different concentrations of glycerol. Reprinted with permission from Ref. [24].
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Imaged CIEF with absorption mode detection is a useful tool for clinical analysis. Quantitation of hemoglobin variants such as A 2 and F may provide diagnostic information of patients with thalassemia syndromes. Analysis of variant A 1c , a glycated form of hemoglobin, is used for long-term monitoring of glycemic control in diabetic patients. All hemoglobin variants can be separated and detected by absorption imaged CIEF. Baseline separation can be achieved by the imaged CIEF instrument for all above mentioned variants, making high precision quantitation for the variants possible. Another example can be found for the analysis of transferrin [25]. Refractive index gradient detection is useful for the analysis of samples lacking tryptophan and / or tyrosine [37], as these compounds do not absorb at 280 nm. Examples are peptide apamin and the peptides from tryptic digests of chicken and bovine cytochrome C. For the imaged fluorescence detector, some standard samples have been tested [13,19]. One of the difficulties in this application is that the heterogeneity of the labelled proteins creates multiple peaks after high-resolution electrophoretic separation. However, the high sensitivity of LIF imaged-CIEF is expected to be valuable, for example, in the investigation of interactions involving proteins, especially for drug discoveries.
7.2. Display of dynamic focusing process One of the advantages of imaged CIEF over single-point detection is that whole column imaging detection monitors the dynamic focusing process as shown in Fig. 8. It
Fig. 8. Dynamic focusing process of two pI markers displayed by imaged CIEF. The concentration of two pI markers are 1 mg / ml. pH 3–10 ampholytes were used, and 1.5 kV voltage was applied.
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provides a direct proof for the computer simulation of the CIEF process [38]. The dynamic focusing information of proteins will be helpful for the identification of proteins and the fundamental investigation of the separation mechanism. Even though the proteins all follow the general focusing rule: focusing toward the pI position from both sides when the samples are distributed uniformly inside the separation column, they have their own separation dynamic focusing patterns. Wu and Pawliszyn [20] first reported the isoelectric focusing process of hemo control ASFC inside a capillary using an absorption imaging detector. The real-time display can tell when the desired separation is achieved and the focusing is complete. This avoids any risk of precipitation due to over-focusing [29].
7.3. Diffusion coefficient measurement Imaged CIEF can be used to measure the diffusion coefficient of proteins [13,16,25]. After normal focusing, a defocusing process is employed. The voltage is turned off, and band broadening due to diffusion can be monitored. The variance of a focused zone with a Gaussian concentration profile can be expressed by the Einstein equation:
s 2 5 2Dt
(5)
where t is defocusing time, s and D are same as that in Eq. (1) and Eq. (2). Therefore, the slope of a plot of spatial variance versus defocusing time yields the diffusion coefficient. The diffusion coefficient of a protein should be strongly correlated to its molecular weight, which will generally allow one to distinguish the separated proteins. Fig. 9 shows the defocusing curves of b-lactoglobulin A (Mw 17 500), myoglobin (Mw 16 700, two variants), and pI marker 8.6 (Mw 509). It is obvious that the pI marker defocused faster than b-lactoglobulin A. Myoglobin did not defocus when it was mixed with the other two compounds. This may be due to precipitation because it does defocus when it was the only component of the sample (data not shown). Therefore, precise measurement of diffusion coefficient is dependent on diluted protein samples. For this reason, a high sensitive imaging detector is favoured for this application, such as LIF imaging using CCD or absorption detection using a PDA sensor.
7.4. Investigation of intermolecular interactions involving proteins In one of the most important applications, imaged CIEF can be used for in vitro observation of the intermolecular interactions involving proteins due to its real-time whole column detection. One example is for monitoring the interaction of iron and bovine transferrin [39]. In this work, iron-free bovine transferrin was first focused inside the column. Then, a plug of ferric ion was introduced into the column from the anodic end. It then travels towards the cathode, and reacts with the iron-free transferrin when it encounters the focused band. The iron-free and iron-complexed transferrins have different pI points, hence, they become focused at different positions within the capillary. The concentration changes of different forms of transferrin during the reaction were monitored by the imaging detector. This interaction can also be monitored by
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Fig. 9. The peak height of b-lactoglobulin, myoglobin and pI marker 8.6 versus defocusing time. They were mixed together and then focused by imaged CIEF for 3.30 min before defocusing.
absorption imaging detector [17]. In another example, interactions between proteins were monitored under CZE mode using an absorption imaging detector [40]. Plugs of two proteins with appropriate pIs were introduced to the separated ends of the capillary by electrokinetic injection. They then traveled towards each other, interacting when they met. The results displayed the dynamic interaction processes. The practical application of the affinity CIEF technique is limited by a low sensitivity due to a non-optimized instrument set-up and a cartridge which is inconvenient for sample introduction. With a well-developed high sensitive absorption or fluorescence detector and a modified cartridge, the association rate and constants of the interaction could be estimated. This technique will find a potential application in biochemical investigation, especially in drug discoveries. Some drugs have specific binding with target proteins. Using a tagging technique, imaged LIF-CIEF will be useful for such a screening job due to its high sensitivity.
8. Conclusion The methodological aspects and instrument set-up for imaging detection have been standardized with the introduction of the commercial instrument with absorption imaging detection. Absorption detection is the most practical and has proven useful for a number of applications. Current research efforts are concentrated on reducing detection noise, enhancing throughput, and improving reproducibility etc. Adopting a suitable lamp, e.g. D 2 and a high well capacity sensor, e.g. PDA, will increase the detection
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sensitivity for absorption imaging. Chip cartridge will allow high throughput and also promises improved imaging capabilities. LIF imaging detection for CIEF performed in a short capillary is likely to be a useful mode for the study of protein chemistry, especially for the interaction investigations, due to its high sensitivity. New design of the commercial instrument will be necessary to facilitate such measurements. Imaged CIEF would benefit from further fundamental research to gain acceptance for additional applications in routine analysis and characterization of biological samples. The dynamic processes, separation patterns, and method development for specific protein analyses are of interest. Additionally, further method development and validation of the application of imaging detection for protein identification are necessary so that this technique can be improved for potential routine analysis. The development of coupling techniques such as LC-CIEF and sampling preparation, e.g. solid phase microextraction (SPME), coupled to imaged CIEF are other promising areas of research projects with improved detection sensitivity.
Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Convergent Bioscience Ltd. We would like to thank Dr Jiaqi Wu for his useful suggestions and Heather Lord for her editorial assistance in preparing this manuscript.
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Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides Qinglu Mao, Janusz Pawliszyn* Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2 L 3 G1 Received 26 November 1998; received in revised form 7 January 1999; accepted 7 January 1999
Abstract Whole column imaging detection has been developed for capillary isoelectric focusing (CIEF) of proteins and peptides. In this imaged CIEF technique, a solution of sample and ampholytes was introduced into a short (4–5 cm), internally coated capillary stabilized by a cartridge. After applying high DC voltage, the isoelectric focusing process takes place and the focused zones are monitored in a real-time mode using the imaging detectors developed. Three types of imaging detectors have been developed including refractive index gradient, laser-induced fluorescence (LIF), and absorption. Of these, absorption imaging detection is the most practical at the present time due to its quantitative ability and universal characteristics. Whole column imaging detection eliminates the mobilization step required for single point detection after the focusing process. Therefore, it provides a fast analysis speed (3–5 min for each sample), and avoids the disadvantages associated with the mobilization process, such as distortion of pH gradient and loss in resolution. In this paper, we review the methodology of imaged CIEF as well as progress in instrumental development, IEF performed on a microchip, and the application to protein and peptide analysis. 1999 Elsevier Science B.V. All rights reserved. Keywords: Capillary isoelectric focusing; Whole column imaging detection; Protein and peptide analysis
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1. Introduction Isoelectric focusing (IEF) is a well known high-resolution technique for biochemical separation [1,2]. It is routinely used for characterization of biological extracts, monitoring protein purification, evaluating the stability or microheterogeneity of protein therapeutics, and determination of protein isoelectric points. Capillary isoelectric focusing (CIEF) [3–7], which was first reported by Hjerten and Zhu [3], combines the high resolving power of conventional gel IEF with the automation and quantitation advantages of capillary electrophoresis (CE) instrumentation. It offers several advantages over traditional gel-based techniques, including ease of automation, quantitation, speed, and detection by UV absorbance (eliminating laborious staining and destaining). In the past decade, CIEF has usually been carried out in commercial CE instruments which have a 20–60-cm long capillary and an on-column UV absorbance detector. Samples, usually proteins, are mixed with carrier ampholytes and introduced into the capillary between the catholyte and anolyte. A high DC voltage is then applied and, as a result, a relatively stable pH gradient is established along the capillary axis by the carrier ampholytes [8]. For a ‘two-step’ mode [4], proteins are first focused at the position where their net charge is zero, i.e. where their isoelectric points (pI values) are the same as the pH values. After focusing, all the focused protein zones are moved through the detection point. Methods using chemical, electrophoretic and hydrodynamic mobilization are well developed. Alternatively, mobilization can be performed during the focusing process with what is called ‘one-step’ CIEF. Proteins are focused and driven towards the detection point by electroosmotic flow (EOF) [9,10]. ‘One-step’ CIEF provides short analysis time, however, it gives lower resolution than mobilization by salt. The use of CIEF for both research and industrial (e.g. quality control, clinical diagnosis) analysis of proteins and peptides is now well established, with reported R.S.D. values for precision in the 2–3% range. The requirement for mobilization to facilitate single-point detection is unique to IEF performed in capillary format. Problems associated with the mobilization process may be encountered, including long analysis time, high risk of protein precipitation, distortion of pH gradient and uneven resolution due to a non-uniform mobilization speed. For example, although the run time of CIEF for a single sample is faster than that of slab gel IEF, CIEF throughput is lower than that with the latter since CIEF is run in a single capillary while slab gel IEF can simultaneously run several samples in different lanes. Also, the conditions and the speed of the mobilization process have to be optimized for different samples to achieve the highest resolution [11]. For these reasons, it is preferable to implement whole capillary imaging detection for CIEF so that the focusing process takes place, as in the slab gel IEF, free from any disturbance, such as the influence of electroosmotic flow (EOF) or hydrodynamic flow. To overcome the drawbacks of single point detection CIEF caused by the mobilization, much effort has been focused on the development of whole column detection for CIEF that eliminates the requirement of mobilization. Whole column detection can be realized by pulling the separation capillary through the detection window [12], however, several limitations are obvious. An additional moving device is required, dynamic noise is relatively high, and distortion of zones still exists due to movement of capillary.
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Another example is a spatial-scanning laser fluorescence detection for CIEF reported by Beale and Sudmeier [13]. This method enables monitoring of the dynamic focusing process, but the slow scan speed (over 15 s for one scan of 8 cm capillary) limits its detection for a fast focusing process (e.g. about 3 min under 600 V/ cm). In the past few years, imaging detection has been found to be ideal for whole column CIEF detection [14]. Three types of imaging detectors have been developed: refractive index gradient (concentration gradient) [15–18], fluorescence [19] and absorption [17,20]. Imaging detection, which provides a real-time monitoring has proven useful in biochemical separation techniques, such as electrophoresis. Two types of imaging detection are used: (1) photograph the focused bands which have luminescent light emission. One well-known example is DNA gel electrophoresis incorporating ethidium bromide [21]. (2) Record transmitted light passing through or fluorescent emission from focused bands. This second type of spatial imaging detection has been developed for capillary electrophoresis. Sweedler and Zare [22] constructed an LIF detector using a charge-coupled device (CCD) camera in a time-delayed integration mode to image the last few centimetres of a separation capillary. Whole column imaging detection for CIEF, known as imaged CIEF, has benefited CIEF analysis significantly. Advances in such imaged CIEF techniques and the performance improvement obtained from the commercial instrument (iCE280, Covergent Bioscience, Etobioke, ON, Canada) have increased its potential application in biotechnology. This review will present a general description on both methodological and application aspects of imaged CIEF technique.
2. Principles of imaged CIEF
2.1. Description With CIEF, IEF is performed in free solution in a capillary format, with the absence of flow. All focused zones are detected by a real-time imaging detector. Unlike that used in slab-gel IEF, where the focused bands are silver-stained and photographed, the principle of imaged CIEF is based on the physical-chemical properties of the focused zones, such as absorption, fluorescence and refractive index gradient (concentration gradient) etc. In single point detection, a detection window is created. In imaged CIEF, an optical system is constructed for whole column detection as shown in Fig. 1. Light from a light source is expanded and projected onto the whole separation capillary, and either transmitted or fluorescence light is detected by a linear or 2-D area sensor, such as CCD and photodiode array (PDA). To simplify the optical alignment, a short separation capillary (5 cm) is favoured. The bare capillary is internally coated in order to minimize electroosmotic flow (EOF) and reduce adsorption of proteins to the column wall. A capillary cartridge is assembled to support the capillary, and is aligned with the imaging detectors. Two pieces of hollow fiber membrane are connected to the capillary ends to isolate the sample solution and the electrolytes, and to allow convenient sample introduction and stable focusing [23]. In imaged CIEF, the pI values of the proteins can be determined directly by their positions in the capillary due to a linear gradient inside
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Fig. 1. Instrument set-up of whole column imaging detection for CIEF. Absorption or refractive index gradient mode with camera (I) placed in the direction of illuminated light; fluorescence mode with camera (II) placed vertically to the direction of illuminated light.
the whole column [24]. A detailed description of the instrument set-up will be presented in Section 3.2 and Section 3.3.
2.2. Resolving power It is a fear that the short capillary may cause a decrease in resolution for imaged CIEF. For this reason, theoretical considerations on the feasibility of a short capillary are addressed here. For a sample zone focused in a capillary by the isoelectric focusing process, concentration has a Gaussian distribution with a variance s [1,2]: C 5 C0 exp(2pEx 2 ) / 2D
(1)
where C5C0 is the maximum concentration, p the mobility slope (2du / dx), E the field strength, D the diffusion coefficient and x position along the capillary.
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]]] D dx s 5 6 ] ]] E (2du)
œ
97
(2)
Using the criterion of three times the variance s for resolved adjacent proteins, the resolving power, DpI, of IEF in terms of s can be expressed as [1]: ]]]]] D d(pH) d(pH) DpI 5 3 ] ]] ]] E (2du) dx
œ
(3)
Eq. (3) shows that good resolution is favoured by a high field strength, a low diffusion coefficient, a high mobility slope dm / d(pH), and a narrow pH gradient. Of the variables, the diffusion coefficient and the mobility slope are intrinsic properties of the analytes, so only pH gradient and the field strength can be varied experimentally. In conventional CIEF, where long capillaries (12–60 cm) are used, pH gradients are more shallow than those seen in imaged CIEF with short capillaries. Satisfactory resolution, however, still can be obtained by adopting a higher field strength. Narrow fused silica capillaries have excellent heat dissipation and so allow high filed strengths (500–800 V/ cm) to be applied. When a narrow pH gradient of 6–8 and a high voltage of 3 kV is used, a resolution of about 0.03 pH units can be achieved with imaged CIEF performed in a 5-cm-long capillary [25]. This is a bit lower than that in optimized conventional single-point detection CIEF with a resolution of 0.01–0.02 pH units, however, the resolution is good enough for clinical analysis. Due to the short capillary, imaged CIEF attains equilibrium faster, e.g. within a few minutes (2–3 min).
3. Method and instrumental development
3.1. Refractive index gradient imaging detector Capillary isoelectric focusing achieves both concentration and focusing of sample components, and so a high concentration gradient is created at each boundary of separated zones inside the capillary. As a result, a refractive index gradient is present at each boundary. The focused zones can therefore be detected through refractive index gradient (concentration gradient) imaging detection. A typical instrument set-up can be constructed on the basis of either the Schileren shadowgraph method [16] or a dark field Toepler–Schliren system [17]. Generally, a well-collimated laser beam is focused onto the capillary column. Theoretically, the laser beam can be regarded as a bundle of infinitesimal light filaments which are parallel. Upon encountering a sample zone inside the capillary, the light filaments are refracted by the refractive index gradient present at each zone boundary, and bent out of their original directions. In the constructed systems, a He–Ne laser (at 633 nm wavelength) [17] or a visible diode laser (at 670 nm) [18] was used as the light source. Compared with the Schileren shadowgraph method, a dark-field Toepler–Schliren system is a more reliable system [17]. The latter is shown in Fig. 2. Its signal is positive and low noise is observed. The light intensity at the maximum deflection angle reaching
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Fig. 2. Instrument set-up of refractive index gradient imaging detector for CIEF based on a dark field Toepler–Schliren system. L1, L2 and L3 are three focusing lenses. Reprinted from Ref. [14] with permission.
the detector can be calculated from the sample concentration, C0 , the capillary length, l, and the zone width, 4s:
S
I0 p B L dn C0 lf I 5 ]] 0.48] ] ]] 2a n dC s 2 8l f
D
(4)
where n is the refractive index of the solution inside the capillary, L the inner diameter of the capillary, and C the protein sample concentration. The signal intensity of the detector is proportional to the refractive index gradient instead of refractive index itself. Refractive index gradient imaging detector is a real-time and universal process. A detection limit of 10 26 M was reached for all proteins tested under optimal conditions.
3.2. Laser-induced fluorescence imaging detector LIF detection is known as one of the most sensitive methods for CE detection [26]. In CIEF, focused protein zones labelled with fluorescent reagent can be detected using a fluorescence imaging detector [19]. As shown in Fig. 1, when the CCD camera is placed in the vertical position to the illumination light direction, fluorescence imaging is
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realized for CIEF. Basically, the laser beam from an argon ion laser was first expanded by either an optical fiber bundle or a microscopic objective lens, and then was focused onto the whole separation column. The fluorescence emission from the focused zones was detected by a CCD camera. A band-pass filter is placed in front of the camera lens to cut off scattering and background light. The wavelength and the band-pass width of the filter depend on the emission spectra of protein samples. The feasibility and performance of the imaged LIF-CIEF system was demonstrated with the proteins b-phycoerythrin, a-D-galactosylated FITC-albumin, insulin-FITC, and casein-FITC. The method is capable of separating and detecting 10 211 M protein samples. Similar LIF imaging detection systems can be found in other examples [13,21,27,28].
3.3. Absorption imaging detector A typical UV-VIS absorption imaging detection system is shown in Fig. 1 where the imaging sensor, camera (I), is fixed linearly to the illumination light direction. A xenon lamp, deuterium lamp, or tungsten lamp can be used as the light source, and a small grating monochromator covering 190–700 nm or a 280 nm bandpass filter can be used to obtain monochromatic light. A fused silica optical fiber bundle guides the light to project onto the CIEF separation column. Between the optical fiber bundle and the capillary column, a cylindrical focusing lens is used. The detector is a camera installed with a linear CCD or PDA. With a 2-D area sensor and a grating, absorption spectra for each focused zone can be obtained [20]. For UV-280 absorption imaging, the typical maximum absorption of proteins, a UV lens is used and the CCD sensor should be UV-sensitive. A capillary cartridge with a 5-cm-long capillary was used to perform CIEF. The absorption imaging mode clearly displays the concentration profiles of the individual focused zones. It is quantitative and universal for most proteins. If the optical alignment for imaged CIEF is optimized, peak broadening will not appear for the imaging of the focused zones after focusing is complete. Absorption imaging detection has become the most practical mode and been well developed. As a result, the commercial instrument is available now. In the following paragraphs, some basic points associated with the absorption imaging detector are presented.
4. Capillary cartridge and imaged CIEF procedures
4.1. Capillary cartridge In the early development stages of imaged CIEF, the cartridge used was very simple: one short capillary (4–5 cm) was connected with two electrolyte reservoirs at the two ends. However, this cartridge is not convenient for sample injection. A new cartridge, as shown in Fig. 3, has been developed for absorption imaging detection [25], and is also suitable for refractive index gradient detection. The separation capillary is internally coated, and its outer polyimide coating is removed. Two pieces of hollow fiber dialysis tubing are glued to the ends of the separation column. Two connection capillaries for sample introduction are glued to the other ends of hollow fiber membranes. Two glass
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Fig. 3. Schematic diagram of the capillary cartridge used for the commercial instrument for absorption imaged CIEF. Electrolyte tanks are affixed to one piece of glass, in which a 5033 mm slash was drilled for UV light to reach the separation column.
plates are used to hold the capillary and one piece of slit is used to cut off the stray light. The membranes that are immersed in the electrolyte reservoirs will allow the small ions like H 1 and OH 2 to pass freely and thus allow CIEF to perform normally. Using such a configuration, the introduction of the sample solutions mixed with carrier ampholytes can proceed without disturbing the electrolytes. In addition, for increasing the throughput, multiple capillaries can be used. A better future option will be the chip cartridge as described later.
4.2. Capillary coating The internal coating of capillary is very important in imaged CIEF. Due to its surface dielectric layers, fused silica capillary usually has a large EOF while a high voltage is applied. If EOF in a capillary is significant, the advantages of imaged CIEF will be lost. To ensure the focused zone remains static after focusing, the inner walls of capillary must be coated. Both static and dynamic coating have proven effective. Several polymer
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coatings provide pH stability and are suitable for CIEF. Examples are polyacrylamide, polyvinylpyrrolidone, polyethylene glycol, poly(vinyl) alcohol and fluorocarbon etc. [6]. Presently, two kinds of coated capillaries are adopted for imaged CIEF [29]. One is chemically coated with linear polyacrylamide [30] and another is physically coated with fluorocarbon. The commercially coated capillary with linear polyacrylamides shows over 100 reproducible runs for imaged CIEF. Fluorocarbon is more robust due to the physical coating and can be used for a long time. To improve performance, conditioning of the capillary is conducted before focusing. Usually, 0.1% methylcellulose solution is forced into the separation column by syringe pump for 20 min. Also, the sample solution is prepared containing 1% methylcellulose to increase the viscosity. With such a treatment, EOF is substantially reduced, and hundreds of runs are observed to be reproducible for the fluorocarbon-coated capillary [29].
4.3. Procedures for imaged CIEF To perform imaged CIEF, the electrolyte reservoirs are first filled with anolyte (H 3 PO 4 solution) and catholyte (NaOH solution), respectively. Then, sample solution is injected into the column cartridge. The commercial imaged CIEF instrument employs an eight port-two position HPLC injection valve for sample injection to the separation column [29]. It has two positions, one is loaded and another is injection. The injection port of the valve is linked to either a needle port for manual sample injection using a micro syringe, or a HPLC autosampler. The sample loop volume is 2.5 ml. The syringe pump is used to deliver sample and washing solution, and is kept running during the entire experiment. The sample injection and the imaged CIEF instrument are fully controlled by a PC computer. After the column is filled with the sample solution, a high voltage (1.5 or 3 kV) is applied to start the isoelectric focusing. The entire process usually takes less than 5 min in a 5-cm-long separation column. A reference image is taken after the sample is filled and stabilised. During the focusing process, images are consecutively taken to monitor the focused zones. Absorption electropherograms are obtained by automatically comparing to the reference image. The final pherogram gives a complete record of the focused zones without mobilization. After one run is finished, the column is washed by the wash solution (0.1% methylcellulose) and the system is ready for the next sample injection. In some cases, protein samples may have a high salt concentration, which compresses the pH gradient and has an impact on the separation pattern. In such case, desalting is necessary to perform using an on-line sample desalting device [23,25].
5. Performance of imaged CIEF
5.1. Reproducibility The reproducibility of imaged CIEF is defined by two parameters: (1) the position inside the column corresponding to pI value; (2) the peak height or peak area for quantitation. Theoretically, the focused zones should be reproducible due to the
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elimination of the mobilization. However, several factors affect the reproducibility, such as electroosmotic flow, hydrodynamic flow and the adsorption of sample to the capillary wall [30]. Electroosmotic flow may become higher after hundreds of runs for a given column since the coating on the inner wall of the column is subject to wear. The electroosmotic flow is usually toward the cathodic end of the column. Also, the solution within the open capillary can be driven by hydrodynamic flow. These factors must therefore be controlled to achieve maximal reproducibility. Fig. 4 shows three consecutive runs for one sample containing five pI markers. For five runs in the experiment, standard deviation of pI marker 8.6 is 0.05 pH units for the pH 2–11 carrier ampholytes, which corresponds to an R.S.D. of 0.6%. Normally, pI markers are added to protein samples as internal standards and the distances between the pI marker and the protein peaks are measured as the protein peak positions (relative positions), instead of the absolute positions along the column. By using the relative position, the standard deviations are less than 0.01 pH units providing R.S.D. of about 0.1%.
5.2. Detection limit CIEF usually concentrates proteins over 100-fold relative to that of the injected sample. Therefore, the detectability for protein should theoretically be higher than that of CE without preconcentration. However, the inability to detect at low UV wavelengths, due to carrier ampholyte absorption, limits the detectability of CIEF with single-point UV detection for analysis of proteins present at concentrations of 5–10 mg / ml or greater [5]. As for absorption imaged CIEF, the detection limit is a bit higher because of the use of CCD sensor. It provides a detection limit of 10 mg / ml for hemoglobin A1 and has a
Fig. 4. Electropherograms for three consecutive runs of five pI markers separated by absorption imaged CIEF. The focusing time was 3.5 min. pH 2–11 ampholytes were used. 0.5% TEMED was added to the sample.
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Fig. 5. Electropherogram of hemo control AFSC (partly glycolyted due to long time storage, indicated by small peaks in the left of four relatively high peaks) separated by imaged CIEF. pH 3–10 ampholytes were used. Left: anode, right: cathode.
baseline noise level of 2310 23 AU when 64 scans of a 2048-pixel linear CCD sensor are averaged as shown in Fig. 5. At high light levels, the shot noise is dominant [31] and mainly limits the detectability. In the commercial version of imaged CIEF, a Xe lamp and a linear CCD are used as light source and sensor, respectively. Due to its low well capacity, CCD is not ideal for absorption imaging detection as it has a very low read noise. Absorption imaging detection is best performed with an array sensor with the ability to discriminate differences of light intensity as small as possible. A specially designed PDA detector usually has a high well capacity of 80–90 M e 2 and makes both read noise and shot noise insignificant. As an example, PDA is recently reported to achieve a low detection limit for absorption imaging detection in CZE, comparable to or exceeding that of a commercial single-point detector [32]. By employing a PDA array and more stable light source, such as a D 2 lamp, instead of the Xe lamp, a lower detection limit would be achieved, thus making absorption imaging detection more comparable to the single-point detection.
6. CIEF performed on chip with imaging detection The microfabrication of analytical instrumentation has received great interest recently, offering a compact, reliable and inexpensive method for chemical and biological separations [33]. CIEF performed on microchip is of interest and is expected to have
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some great advantages over the conventional CIEF techniques in separation speed, compact size, integration and throughput etc. The feasibility of isoelectric focusing (IEF) performed on chip was demonstrated for the first time via absorption imaging detection in our group (Mao and Pawliszyn, submitted). Absorption imaging detection is ideal for chip IEF due to its universal and spatial detection mode. In most CE systems, laser-induced fluorescence is widely used, although limited by the need for labelling of proteins lacking fluorescence. Direct single point UV absorbance detection also is not suitable for chip IEF due to the requirement for mobilization. Instead of using the capillary cartridge, a chip cartridge for IEF was fixed in the optical path. Chip channels for separation were fabricated on a piece of quartz microchip using photolithography and chemically etching process (Albert Microelectronic Corporation, Edmonton, Alberta, Canada). The separation channel is 40 mm long, 100 mm wide and 10 mm deep, and was coated with linear polyacrylamide to reduce electroosmotic flow and decrease the adsorption of proteins to channel walls. Low molecular mass pI markers, substituted monomethylphenols, and the protein pI marker, myoglobin, were selected as model samples. Good performance of IEF has been achieved in microchip channels as shown in Fig. 6. The focusing process is reproducible and the resolution is similar to that of imaged CIEF in capillary format. Chip cartridge IEF has some advantages over the capillary cartridge one. It offers a smooth baseline with less spike noise and eases the optical alignment between the channel and slit due to the square geometry of the separation channel. Also, multiple channels can be more easily constructed to increase throughput than in a capillary cartridge [20,34].
Fig. 6. Separation of pI markers 7.4 and 6.6 on coated channel with chip cartridge. Concentration of each component is 4 mg / ml; pH 3–10 ampholytes were used and a high voltage of 3 kV was applied. Left: cathode, right: anode.
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7. Applications
7.1. Analysis of proteins and peptides The direct application of imaged CIEF is to analyze proteins and peptides. Many protein standard samples have been analyzed using absorption imaging detection. Examples are myoglobin, hemoglobin, cytochrome c, anti-a-acid glycoprotein, IgG, a-acid glycoprotein and monoclonal anti-a-antitrypsin transferrin [29,35,36]. Satisfactory separations have also been achieved for a number of real samples (unpublished data). Peptides can also be analyzed by imaged CIEF [29]. At 280 nm, only tyrosine and tryptophan have absorption, and sensitivity of UV absorption detector is low for many peptides. Fig. 7 shows an electropherogram of a peptide which contains only one tyrosine and no tryptophan. For this reason, a high concentration (2 mg / ml) was adopted for the analysis. However, due to the peptide being concentrated in the focusing, the peptide easily precipitates in less than 2 min after the focusing starts. By adding glycerol to the sample, its solubility can be enhanced and the focusing becomes possible, as shown in Fig. 7. Reproducible results can be achieved when 20% glycerol is added to the sample.
Fig. 7. Separation of peptides (Ac-Asp-Asn-pTyr-Ile-Ile-Pro-Leu-Pro-Asp-Pro-Gly-OH) by absorption imaged CIEF under different concentrations of glycerol. Reprinted with permission from Ref. [24].
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Imaged CIEF with absorption mode detection is a useful tool for clinical analysis. Quantitation of hemoglobin variants such as A 2 and F may provide diagnostic information of patients with thalassemia syndromes. Analysis of variant A 1c , a glycated form of hemoglobin, is used for long-term monitoring of glycemic control in diabetic patients. All hemoglobin variants can be separated and detected by absorption imaged CIEF. Baseline separation can be achieved by the imaged CIEF instrument for all above mentioned variants, making high precision quantitation for the variants possible. Another example can be found for the analysis of transferrin [25]. Refractive index gradient detection is useful for the analysis of samples lacking tryptophan and / or tyrosine [37], as these compounds do not absorb at 280 nm. Examples are peptide apamin and the peptides from tryptic digests of chicken and bovine cytochrome C. For the imaged fluorescence detector, some standard samples have been tested [13,19]. One of the difficulties in this application is that the heterogeneity of the labelled proteins creates multiple peaks after high-resolution electrophoretic separation. However, the high sensitivity of LIF imaged-CIEF is expected to be valuable, for example, in the investigation of interactions involving proteins, especially for drug discoveries.
7.2. Display of dynamic focusing process One of the advantages of imaged CIEF over single-point detection is that whole column imaging detection monitors the dynamic focusing process as shown in Fig. 8. It
Fig. 8. Dynamic focusing process of two pI markers displayed by imaged CIEF. The concentration of two pI markers are 1 mg / ml. pH 3–10 ampholytes were used, and 1.5 kV voltage was applied.
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provides a direct proof for the computer simulation of the CIEF process [38]. The dynamic focusing information of proteins will be helpful for the identification of proteins and the fundamental investigation of the separation mechanism. Even though the proteins all follow the general focusing rule: focusing toward the pI position from both sides when the samples are distributed uniformly inside the separation column, they have their own separation dynamic focusing patterns. Wu and Pawliszyn [20] first reported the isoelectric focusing process of hemo control ASFC inside a capillary using an absorption imaging detector. The real-time display can tell when the desired separation is achieved and the focusing is complete. This avoids any risk of precipitation due to over-focusing [29].
7.3. Diffusion coefficient measurement Imaged CIEF can be used to measure the diffusion coefficient of proteins [13,16,25]. After normal focusing, a defocusing process is employed. The voltage is turned off, and band broadening due to diffusion can be monitored. The variance of a focused zone with a Gaussian concentration profile can be expressed by the Einstein equation:
s 2 5 2Dt
(5)
where t is defocusing time, s and D are same as that in Eq. (1) and Eq. (2). Therefore, the slope of a plot of spatial variance versus defocusing time yields the diffusion coefficient. The diffusion coefficient of a protein should be strongly correlated to its molecular weight, which will generally allow one to distinguish the separated proteins. Fig. 9 shows the defocusing curves of b-lactoglobulin A (Mw 17 500), myoglobin (Mw 16 700, two variants), and pI marker 8.6 (Mw 509). It is obvious that the pI marker defocused faster than b-lactoglobulin A. Myoglobin did not defocus when it was mixed with the other two compounds. This may be due to precipitation because it does defocus when it was the only component of the sample (data not shown). Therefore, precise measurement of diffusion coefficient is dependent on diluted protein samples. For this reason, a high sensitive imaging detector is favoured for this application, such as LIF imaging using CCD or absorption detection using a PDA sensor.
7.4. Investigation of intermolecular interactions involving proteins In one of the most important applications, imaged CIEF can be used for in vitro observation of the intermolecular interactions involving proteins due to its real-time whole column detection. One example is for monitoring the interaction of iron and bovine transferrin [39]. In this work, iron-free bovine transferrin was first focused inside the column. Then, a plug of ferric ion was introduced into the column from the anodic end. It then travels towards the cathode, and reacts with the iron-free transferrin when it encounters the focused band. The iron-free and iron-complexed transferrins have different pI points, hence, they become focused at different positions within the capillary. The concentration changes of different forms of transferrin during the reaction were monitored by the imaging detector. This interaction can also be monitored by
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Fig. 9. The peak height of b-lactoglobulin, myoglobin and pI marker 8.6 versus defocusing time. They were mixed together and then focused by imaged CIEF for 3.30 min before defocusing.
absorption imaging detector [17]. In another example, interactions between proteins were monitored under CZE mode using an absorption imaging detector [40]. Plugs of two proteins with appropriate pIs were introduced to the separated ends of the capillary by electrokinetic injection. They then traveled towards each other, interacting when they met. The results displayed the dynamic interaction processes. The practical application of the affinity CIEF technique is limited by a low sensitivity due to a non-optimized instrument set-up and a cartridge which is inconvenient for sample introduction. With a well-developed high sensitive absorption or fluorescence detector and a modified cartridge, the association rate and constants of the interaction could be estimated. This technique will find a potential application in biochemical investigation, especially in drug discoveries. Some drugs have specific binding with target proteins. Using a tagging technique, imaged LIF-CIEF will be useful for such a screening job due to its high sensitivity.
8. Conclusion The methodological aspects and instrument set-up for imaging detection have been standardized with the introduction of the commercial instrument with absorption imaging detection. Absorption detection is the most practical and has proven useful for a number of applications. Current research efforts are concentrated on reducing detection noise, enhancing throughput, and improving reproducibility etc. Adopting a suitable lamp, e.g. D 2 and a high well capacity sensor, e.g. PDA, will increase the detection
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sensitivity for absorption imaging. Chip cartridge will allow high throughput and also promises improved imaging capabilities. LIF imaging detection for CIEF performed in a short capillary is likely to be a useful mode for the study of protein chemistry, especially for the interaction investigations, due to its high sensitivity. New design of the commercial instrument will be necessary to facilitate such measurements. Imaged CIEF would benefit from further fundamental research to gain acceptance for additional applications in routine analysis and characterization of biological samples. The dynamic processes, separation patterns, and method development for specific protein analyses are of interest. Additionally, further method development and validation of the application of imaging detection for protein identification are necessary so that this technique can be improved for potential routine analysis. The development of coupling techniques such as LC-CIEF and sampling preparation, e.g. solid phase microextraction (SPME), coupled to imaged CIEF are other promising areas of research projects with improved detection sensitivity.
Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Convergent Bioscience Ltd. We would like to thank Dr Jiaqi Wu for his useful suggestions and Heather Lord for her editorial assistance in preparing this manuscript.
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