Capillary Isoelectric Focusing with Laser-Induced Fluorescence Whole Column Imaging Detection as a Tool To Monitor Reactions of Proteins Zhen Liu and Janusz Pawliszyn* Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Received November 26, 2003
Capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) has the characteristics of high resolution, high speed and high sensitivity for separation of amphoteric biomolecules. These features enable a CIEF-LIF-WCID system to monitor the dynamic process of a protein reaction. The reaction can be a physical change or a chemical reaction, provided that the kinetics of the reaction is slower than the focusing speed or that the intermediates involved have long enough life-span compared to the analysis time. The processes of denaturation (a physical reaction), reduction and carbamylation (both chemical reactions) were dynamically monitored. The CIEF profiles at successive reaction times clearly displayed the formation of different products at different stages. At incomplete denaturation, intermediates with higher apparent pI values relative to the products at complete denaturation were detected. Carbamylation products of a protein were detected when the protein reacted with a urea solution that had prepared three months earlier, exhibiting gradually decreased pI values. Mechanisms involved in these reactions were rationalized. A combined mechanism of denaturation and reduction was suggested to explain the denaturing process under high concentrations of urea. Potential applications and critical factors to manipulate these reactions were also discussed. Keywords: capillary isoelectric focusing • laser-induced fluorescence • whole column imaging detection • reduction • denaturation • carbamylation
Introduction As a capillary-format of isoelectric focusing (IEF), capillary isoelectric focusing (CIEF) is a high-resolution electrophoretic technique for the separation of proteins and other amphoteric biomolecules.1-5 Analytes are separated based on their difference in isoelectric points (pI). As the sample components are focused into narrow bands, CIEF also acts as a sample concentration technique. CIEF has been widely used for analytical (the analytes include proteins,6,7 peptides,8,9 antibodies,10,11 viruses12,13 and cell14) and preparative15-17 purposes. This technique is expected to play an increasingly important role in the rapidly developing field of proteomics. Whole-column imaging detection (WCID)18-24 is a novel detection scheme in CIEF, which eliminates the mobilization step required by the conventional single point detection.25-30 CIEF-WCID has several advantages over single point detection. First, the inherent characteristics of CIEF (high efficiency and high resolution) are maintained. Second, the optimization of separation conditions is simplified and the total separation time is greatly reduced. Generally, separation can be completed within 5 min. Three types of detectors have been employed for WCID, including concentration gradient,18-21 UV adsorption,20,22 and laser-induced fluorescence (LIF).23,24 Recently, we31 * To whom correspondence should be addressed. Phone: (519) 888-4641. Fax: (519) 746-0435. E-mail:
[email protected]. 10.1021/pr034114w CCC: $27.50
2004 American Chemical Society
developed a CIEF system with a liquid core waveguide (LCW)LIF-WCID. The sensitivity was enhanced by 3-5 orders of magnitude compared with UV-WCID. Due to the improved sensitivity, isoelectric precipitation,32 a main drawback in IEF which is more serious at higher concentration, is effectively prevented by using lower sample concentration. CIEF-WCID has been employed in the analysis of proteins,33,34 peptides,34,35 antibodies,33,36 and viruses.37 Further, because of its real-time display ability, CIEF-WCID has found particular applications in monitoring fast dynamic processes such as metal-protein interaction,38 protein-protein interaction39 and immunoreaction.40 The monitoring applications of CIEF-WCID can be expanded to a reaction with slow kinetics. The pI value of a protein is a parameter determined by its compositional and structural factors such as amino acid composition, conformation and post-translational modification. Many biological reactions of proteins involve compositional change, conformational variation and post-translational modification, resulting in differences in pI. Therefore, CIEF can be used to monitor reactions provided that the kinetics are slower than the analysis speed or that the intermediates involved have long enough lifetime compared with the analysis time. Such a reaction can be a chemical reaction, a physical changes or a combination of these; for example, thiol redox reactions, NH2 terminus blocking, deamidation, folding/unfolding, and so on. In principle, Journal of Proteome Research 2004, 3, 567-571
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CIEF-UV-WCID can be used to monitor a wide variety of reactions due to the universality of UV absorption detection. However, when the biological species of interest have poor solubility, the associated precipitation problem will hamper the employment of UV-WICD. In such a case, LIF-WCID is a good alternative if the analytes are fluorescent or fluorescently labeled, providing clear focusing profiles. Compared to other methods such as spectrophotometry,41,42 gel electrophoresis43 and high-performance liquid chromatography,44 CIEF-LIFWCID has advantages of lower sample consumption and higher speed. Denaturation (a physical change of conformation), reduction and carbamylation (both chemical reactions) are of importance in protein chemistry and technology. Studies of denaturation and reduction can provide additional information on the structure, properties, and function of proteins. These two kinds of processing are widely used in protein analysis methods such as 2D gel electrophoresis, to break macromolecular interactions and to solubilize proteins in the gel.45 Urea (one of the most widely used denaturants) and dithiothreitol (DTT, a typical reducing agent) are often added to the 2D separation medium. Carbamylation is a typical post-translational processing of protein.46 In clinical biochemistry, carbamylation of hemoglobin and plasma proteins has been shown to be related to uremia.47 In denaturing IEF48 and peptide mapping,49 unwanted carbamylation gives rise to artifacts. In this study, the three reactions were dynamically monitored by using the CIEF-LCWLIF-WCID system. Different products at different reaction times were detected, which offers an insight into the mechanisms of these reactions. Potential application and theoretical guidelines implicated from this study are discussed.
Materials and Methods Apparatus. The CIEF-LCW-LIF-WCID instrument setup used was built in-house as described previously,31 except that a ventilating fan and two ventilating holes were built onto the black box. The working temperature of the CCD camera was set at -30 °C. CIEF cartridges with separation channels of 5.0 or 7.8 cm long (effective length) and 167 µm ID Teflon AF 2400 capillary were used. Reagents and Materials. Polyvinylperrolidone (PVP, average molecular weight about 360 000 and intrinsic viscosity 80-100 K), fluorescein isothiocyanate (FITC)-insulin, and urea were obtained from Sigma (St Louis, MO). Green fluorescent protein (GFP) at 0.5 mg/mL and DTT were donated by Convergent Bioscience Ltd (Toronto, Canada). Carrier ampholytes were Pharmalytes (pH 3-10) from Sigma. Anolyte and catholyte were 100 mM phosphoric acid and 100 mM sodium hydroxide, respectively. Water was purified with an ultrapure water system (Barnstead/Thermolyne, Dubuque, IA), and was used to prepare all solutions. Optical fiber with 100 µm core was purchased from Polymicro Technologies Inc (Phoenix, AZ). Teflon AF 2400 capillary of 167 µm ID and 364 µm OD was purchased from Random Technologies (San Diego, CA). Urea Solution Preparation. For fresh urea solutions, a certain amount of urea was mixed with certain volumes of the protein to be investigated and other constituents such as carrier ampholytes and PVP, and then diluted to desired volume with water. For old urea solutions, a stock solution of 10 M urea, which has been stored at room temperature for three months, was in place of the solid urea and the preparation procedure was the same as for fresh urea solutions. 568
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Figure 1. CIEF of FITC-insulin (A) and GFP (B) under native conditions. Separation channel: 5 cm (A) or 7.8 cm (B) × 167 µm ID Teflon AF 2400 capillary; sample: 2 µg/mL FITC-insulin (A) or 0.5 µg/mL GFP (B) containing 2% Pharmalytes (pH 3-10), 0.5% PVP; applied voltage: 3 kV; excitation wavelength: 488 nm; detection filter: 530 nm long-pass filter.
CIEF. Before separation, the separation capillary was conditioned with water and 0.5% PVP aqueous solution for 30 min for each. The samples were prepared by mixing the protein with a certain amount of DTT or urea along with other constituents such as carrier ampholytes, PVP and water, and then stored in a syringe. The sample was injected into the separation channel at certain times, and then CIEF was performed. Imaging detection was carried out at desired times. The focusing process was finished within 10 min.
Results and Discussion CIEF under Native Conditions. For comparison of the focusing profiles before and after initiating the reactions to be monitored, CIEF of FITC-insulin and GFP under native conditions (i.e., in the absence of DTT and urea) was carried out and the electropherograms are shown in Figure 1. Although GFP was focused very well, FITC-insulin exhibited two broad peaks. Previously, the broad peaks of FITC-insulin were ascribed to the interaction of the protein with the capillary wall.31 However, multiple labeling seems to be the main reason. Insulin is composed of two different subunits, the A chain and B chain, which are cross-linked by two interchain disulfide bonds. The A chain contains an intrachain disulfide bond. Insulin contains three amine groups, one -amine on the single lysine group on the B chain and two R-amines at the two N-terminals. FITC is an amine-reactive dye, reactive to the -amine or R-amine or both, depending on the conditions used for labeling. Generally, for a molecule with n amino groups, 2n - 1 reaction products are possible.50 Therefore, if FITC moieties are attached to the three amines of insulin, the labeling procedure could generate up to 7 products. As the 7 products maybe have so similar pI values that they could not be
Monitoring Reactions of Proteins
Figure 2. CIEF of FITC-insulin at successive reducing times. Sample: 2 µg/mL FITC-insulin containing 2% Pharmalytes (pH 3-10), 0.5% PVP, 50 mM DTT; other conditions as in Figure 1B; *, background signal.
completely resolved under the CIEF conditions used, one or broad peaks could be observed. Reduction. In a reduction reaction of protein, the disulfide bonds in a protein molecule are broken by a reducing reagent. For a single-chain protein, only one product will be generated. For a protein with multiple chains that are linked through disulfide bridges, multiple products will be observed. The CIEF electropherograms of FITC-insulin at successive reducing times are shown in Figure 2. In the initial 135 min, the focusing patterns varied noticeably, whereas the focusing patterns were quite similar at 165 and 195 min. This means that the composition in the reaction mixture changes significantly during the initial stage of reaction but becomes constant when the reaction is close to completion. At incomplete reduction, particularly at 45 min, at least 5 peaks were observed. However, the number of peaks reduced after the reduction was complete; only 4 peaks were observed at 165 and 195 min. The 4 peaks were most likely the reduction products of FITC-insulin. As mentioned above, FITC multiply labeled insulin could generate up to 7 products. After the disulfide bonds have been broken, the 7 products are cleaved into 4 fragments, namely the R-labeled A chain, R-labeled B chain, -labeled B chain, and R,-labeled B chain. Comparing Figure 2 with Figure 1A, the peak efficiency was found to be greatly improved with the use of reducing conditions. Under native conditions, the incomplete separation of the multiply labeled products resulted in broad peaks, whereas under reducing conditions, each reduction product gave an individual peak, exhibiting higher peak efficiency. On the basis of the above discussion, it is clear that, in any application where protein reduction is involved, sufficient reaction time is critical to obtain reliable results. Besides, reducing CIEF can be suggested to verify whether a labeling procedure results in single or multiple labeling. In the field of bioconjugate chemistry, the efficiency of a labeling procedure is characterized by the degree of labeling, usually expressed as the dye/protein (D/P) ratios. According to the vendor, the D/P ratio for the FITC-insulin used in this study was 1.2:1. In fact, the degree of labeling has no relationship with single labeling. Even at a stoichiometric ratio of 1:1, multiple labeling is still possible. If the protein contains several subunits linked with disulfide bonds, then reducing CIEF should be a reliable method for purity testing of labeled products. Denaturation. Urea can break most nonconvalent interactions in the protein molecule, including ionic bonds, hydrogen bonds and hydrophobic interactions.51 Unlike reduction,
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Figure 3. CIEF of GFP at successive denaturing times. Sample: 0.5 µg/mL GFP containing 2% Pharmalytes (pH 3-10), 0.5% PVP, 4 M urea; other conditions as in Figure 1B.
Figure 4. CIEF of FITC-insulin at successive denaturing times. Sample: 2 µg/mL FITC-insulin containing 2% Pharmalytes (pH 3-10), 0.5% PVP, 7 M urea; other conditions as in Figure 1B.
which breaks covalent S-S bonds, denaturation only changes the conformation of the protein. GFP is a single-chain, neturally flurorescent protein. Due to its very compact cylindrical structure with the fluorophore centrally located in the molecule, GFP is very resistant to denaturation.51 Figure 3 shows the CIEF patterns of GFP at two denaturing times in the presence of 4 M urea. It is clear that an intermediate with a high apparent pI value was produced during the process of denaturation, which seems to be a protein-urea adduct or complex. After the denaturation process was finished, a triple peak was observed, located in the same position as it was in the absence of urea (see Figure 1B). As the protein-urea complex showed a higher apparent pI than the protein did under the complete denaturation state, the denaturation time is critical for reliability. In the literature on 2D gel electrophoresis, denaturing gel IEF and CIEF, little attention was paid to the denaturation time. As with the reduction reaction, insufficient denaturation is less apparent in denaturing 2D gel electrophoresis and denaturing gel IEF due to their long separation times and continual presence of denaturing conditions. However, for a high-speed monitoring method, proteins must be completely denatured before experiment. The denaturation process of FITC-insulin was also monitored, and the focusing patterns at three reaction times are shown in Figure 4. At 60 min, some intermediate peaks, located at higher pH range relative to the products at complete denaturation, were observed. This follows the same trend seen in Figure 3 for GFP. At 120 min, seven peaks were observed, the peak efficiency was improved, and the peaks shifted toward the acidic pH range. At 180 min, the peak efficiency was further improved, the peaks were slightly shifted to the anode end, and only four peaks were observed. By comparing Figure 4 and Journal of Proteome Research • Vol. 3, No. 3, 2004 569
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Figure 5. CIEF of GFP at successive reaction times with aged 6 M urea solution. Sample: 0.5 µg/mL GFP containing 2% Pharmalytes (pH 3-10), 0.5% PVP, 6 M urea; applied voltage: 0-5 min, 1 kV; after 5 min, 3 kV; other conditions as in Figure 1A.
Figure 1A, it can be seen that the peak efficiency was greatly improved under denaturing conditions. The increased peak efficiency indicates that the homogeneity of the peaks increased due to processing with urea. The number of FITC-insulin peaks seen under denaturing conditions cannot be explained theoretically according to the usual mechanism of denaturation. If urea did not break the disulfide bonds, then the CIEF pattern should have the same number of peaks under denaturing conditions as it has under native conditions. However, the number of peaks seen under complete denaturation was the same as under complete reduction. A possible explanation is that a reducing mechanism was involved during the denaturation of FITC-insulin. Such reasoning is supported by the viewpoint in the literature that a concentrated urea solution may bring about rupture of some disulfide bridges.52 As the number of the disulfide bonds in the insulin molecule is small, 7 M urea seems strong enough to break all the disulfides, resulting in four peaks. Thus, the mechanism involved in ureatreatment of FITC-insulin could be a combination of denaturation and reduction. To verify such a hypothesis, CIEF experiments with UV-WCID of nonlabeled insulin were performed under native, reducing and denaturing conditions. Unfortunately, due to the poor solubility of insulin under the conditions used and the poor UV detection sensitivity, no clear focusing profiles were obtained at a low concentration of insulin while precipitation occurred severely at a high concentration. Carbamylation. A typical carbamylation protocol involves treatment of protein with a concentrated urea solution at neutral to alkaline pH for increasing lengths of time.47 In solution, urea is in equilibrium with ammonium cyanate and the level of the later increases with increasing temperature and pH. An initially cyanate-free urea solution may become a cyanate-rich solution after extended storage. Cyanate is very reactive toward primary amines in proteins, and the carbamylated amines (N-terminus and -amine of lysine) are relatively stable over a wide pH range.49 When a protein is carbamylated, its pI becomes more acidic because positive charges on the parent molecule are depleted. As the number of the cyanates reacted with an identical protein molecule increases, the pI value of the carbamylated protein gradually decreases. Carbamylation was carried out using a 6 M urea solution diluted from a 10 M urea solution that had been stored at room temperature for three months. Figure 5 shows the focusing patterns of GFP at successive reaction times with 6 M urea in the CIEF separation solution. The CIEF pattern of GFP carbamylated using freshly prepared 6 M urea at a reaction time of 30 min is shown in Figure 6 for comparison. When using 570
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Figure 6. CIEF of GFP under treatment with fresh 6 M urea. Sample: 0.5 µg/mL GFP containing 2% Pharmalytes (pH 3-10), 0.5% PVP, 6 M urea; other conditions as in Figure 5.
the fresh urea solution, the denaturation process was completed very quickly, and no intermediate or product peak was observed except the peaks for GFP itself. The CIEF profile kept nearly the same as the reaction time was extended up to several hours. As a contrast, when using the old urea solution of identical concentration, denaturation-induced intermediates and carbamylation adducts were detected. At 45 min (Figure 5), some intermediates with high pI values were observed, which is in good agreement with the sole denaturing mechanism discussed above. Within 75-135 min, carbamylation products were observed, which have more acidic pI values compared to the parent protein; along the reaction time proceeding, the carbamylated proteins further shifted to the acidic end. At 165 min, all the carbamylated proteins disappeared from the separation channel, as they gained a pI value lower than the lowest pH (pH 3) of the pH gradient formed inside the separation channel. In denaturing IEF, use of fresh urea solution is usually emphasized in the literature.48,52 Due to its slow focusing speed, gel-IEF is unable to detect the formation of carbamylation adducts. With the CIEF-LCW-LIFWCID, carbamylation adducts caused by an un-fresh urea solution were observed in this study. Advantages and Disadvantages. The CIEF-LIF-WCID method has several advantages for monitoring protein reactions. First, the combination of CIEF and LIF detection offers high selectivity. In CIEF, only amphoteric molecules such as proteins and peptides are focused, avoiding interference from nonamphoteric molecules. The LIF detection eliminates interference and background from nonfluorescent molecules. Second, the employment of LIF detection provides high sensitivity, which is enhanced by the concentration effect of CIEF. In this study, only 1-4 nanograms of protein were needed for a single run, providing detection signals of hundreds times higher than the noise level. In contrast, milligram amounts of protein are required in spectrophotometric methods41,42 and micrograms in gel electrophoresis43 and high-performance liquid chromatography.44 Last, this method offers high speed. In this study, only 10 min was needed for a single analysis. By using a capillary of smaller inner diameter and higher voltage, the separation time can be further reduced. In comparison, several hours are required in spectrophotometric methods.41,42 The CIEF-LIF-WCID method also has some disadvantages. The protein to be studied must be fluorescent, which involves timeconsuming labeling procedures for nonfluorescent proteins. Also, the LIF detection fails to detect nonfluorescent products that, in some applications, may be of interest.
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Monitoring Reactions of Proteins
Conclusions The CIEF-LIF-WCID system has been shown to be a useful tool for monitoring the dynamic processes of reduction, denaturation and carbamylation of proteins. This study shed light on the possible mechanisms of these reactions and their potential applications, and offered theoretical guidelines for the use of these reactions in related fields. Reaction time was found to pay a critical role for the reliability of the results of reduction and denaturation, especially in a high-speed analytical method such as CIEF. The method presented in this work can be further applied to other types of post-translational processing and chemical modifications if the kinetics are slow enough compared to the separation speed. This method proved to be of high efficiency, high selectivity, high speed, and high sensitivity.
Acknowledgment. The authors gratefully thank Convergent Bioscience Ltd for donation of the reagents GFP and DTT. J.P. would like to acknowledge the Natural Sciences and Engieering Research Council of Canada (NSERC) and Canada Research Chair (CRC) for fundings.
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