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mauro, O. Zerbinati, M.L. Tosato, Environ. Sci. Technol. 24 ( 1990 ) 1559. [ 28 ] M. Bideau, B. Claudel, L. Faure, M. Rachimoellah, J. Photochem. 39 ( 1987 ) 107. [ 29 ] J.J. Pignatello, Environ. Sci. Technol. 26 ( 1992 ) 944. [ 30 ] M. Barbeni, C. Minero, E. Pelizzetti, E. Borgarello, N. Serpone, Chemosphere 16 ( 1987 ) 2225. [ 31 ] D.L. Sedlak, A.W. Andren, Environ. Sci. Technol. 25 ( 1991 ) 777. [ 32 ] C. Maillard, Ch. Guillard, P. Pichat, Chemosphere 24 ( 1992 ) 1085. [ 33 ] M. Halmann, J. Photochem. Photobiol. A Chem. 66 ( 1992 ) 215. [ 34 ] A. Mills, R. Davies and D. Worsley, Chem. Soc. Rev., ( 1993 ) 417.
[ 35 ] C.K. Graëtzel, M. Jitousek, M. Graëtzel, J. Mol. Catal. 60 ( 1990 ) 375. [ 36 ] C.S. Turchi, D.F. Ollis, J. Catal. 122 ( 1990 ) 178. [ 37 ] T. Hisanaga, K. Harada, K. Tanaka, J. Photochem. Photobiol. A Chem. 54 ( 1990 ) 113. [ 38 ] E. Pramauro, M. Vincentini, V. Augugliaro, L. Palmisano, Environ. Sci. Technol. 27 ( 1993 ) 1790. [ 39 ] G. Penìuela and D. Barceloè, J. Chromatogr. A 823 ( 1998 ) 81. [ 40 ] C.H. Walling, Acc. Chem. Res. 8 ( 1975 ) 125. [ 41 ] W.G. Barb, J.H. Baxendale, P. George, K.R. Hargrave, Trans. Faraday Soc. 47 ( 1951 ) 591. [ 42 ] G.K. Pandit, S. Pal, A.K. Das, J. Agric. Food Chem. 43 ( 1995 ) 171.
Fluorescent derivatization for low concentration protein analysis by capillary electrophoresis Peter R. Banks*
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Blvd. W., Montreal, Que. H3G 1M8, Canada Fluorescence derivatization can allow for the low concentration analysis of proteins by capillary electrophoresis. Major problems arising from inef¢cient chemistry and multiple derivatives must be overcome, however, for the method to be successful. A number of methods are discussed in this review. z1998 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence; Derivatization; Protein; Capillary electrophoresis
1. Introduction Although capillary electrophoresis ( CE ) is a relatively new separation technique when compared to more established methods such as high performance liquid chromatography ( HPLC ), gas chromatography and slab gel electrophoresis, its numerous advantages *Fax: +1 (514) 848-2868. E-mail: [email protected]
have resulted in many diverse research efforts and widespread use. These advantages include high separation ef¢ciency, analysis speed, instrumentation simplicity and the ability to perform unique analyses, such as the examination of the contents of a single cell [ 1 ]. Furthermore, inferior analytical performance due to both poor migration time and injection reproducibility has been overcome by the advent of new commercial instrumentation where reproducibilities below 1% RSD are now standard [ 2 ]. Despite these advantages and advances, detection is still a problem for analyses requiring the detection of low concentration analyte. The primary focus of my laboratory is on low concentration protein analysis by capillary electrophoresis, thus detection is one of the main problems I experience in my research efforts. The ubiquitous deuterium lamp-based UV absorbance detector is unsuited for low concentration protein analysis since the short path length ( approx. 39 Wm for a 50 Wm I.D. capillary ) of the detection cell limits the determination of proteins to concentrations typically greater than micromolar (WM ) levels. Even with extended path length devices such as a bubble cell [ 3 ], Z-cell [ 4 ] or high sensitivity cell [ 5 ], limits of detection ( LOD )
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Fig. 1. Derivatization of a lysine residue in a protein with FITC to produce a £uorescein thiocarbamyl derivative.
are only extended by one order of magnitude, at best. Thus I ( and others ) investigate alternative detection methods which can provide lower LOD. An obvious candidate for low concentration detection is £uorescence, especially when the excitation source is a laser. Proteins are intrinsic £uorophores if they contain aromatic amino acid residues. Both excitation and emission are in the UV, however, which limits the type of laser that can be used. Swaile and Sepaniak have used a frequency doubled Ar laser ( 257 nm ) [ 6 ], Lee and Yeung used the 275.4 nm line from a similar laser [ 7 ], and a number of groups, including mine [ 8 ], have used a KrF excimer laser ( 248 nm ) for the detection of proteins separated by CE. Systems using these lasers can generally provide nanomolar ( nM ) LOD for proteins. Unfortunately, these UV lasers tend to be expensive compared to lasers that operate in the visible. The Ar lasers used for native protein detection must be water-cooled to generate mW UV power necessary for optimal £uorescence generation. These lasers are about an order of magnitude more expensive than their air-cooled cousins which produce mW power levels in the visible ( 488 nm ). Even the KrF laser is about three times more expensive than this laser.
For this reason, most laser-induced £uorescence ( LIF ) detectors for CE separations use inexpensive lasers that operate in the visible or near UV. The Ar laser operating at 488 nm is the most common, but other lasers used include the UV line of the He Cd laser ( 325 nm ), the green He Ne laser ( 543.5 nm ) and increasingly, very inexpensive diode lasers operating in the red ( i.e. 635 nm ). LIF detection with visible lasers is also available in commercial instrumentation: for example, Beckman instruments offers excitation at both 488 and 635 nm in their P / ACE systems. Few native proteins contain a £uorophore which can be excited by these lasers. Yet proteins can be converted into suitable £uorescent derivatives by chemical derivatization, typically using amine-reactive £uorescent or £uorogenic probes. These probes contain an amine-reactive moiety or are converted into a £uorophore by their covalent attachment to proteins through primary amines, such as the N-terminus or lysine residues. This process is demonstrated in Fig. 1 for the derivatization of a lysine residue in a protein with the most commonly used £uorescent derivatizing reagent, £uorescein isothiocyanate ( FITC ). Using this probe, picomolar ( pM ) concentrations of £uorescein
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thiocarbamyl ( FTC )-amino acids have been detected [ 9 ].
2. Problems in the £uorescent derivatization of proteins 2.1. Probe concentration requirements
In order to detect picomolar concentrations of FTCamino acids, they must ¢rst be derivatized at high concentration ( typically 1034 M ) then diluted by a factor of 108 . The high amino acid concentration for the derivatization reaction is required because the chemistry of FITC derivatization of primary amines is slow and inef¢cient. My group has demonstrated that the lowest concentration of amino acid that can be recognizably derivatized is about 1036 M [ 10 ]. This limitation is also apparent for protein derivatization. Thus the LOD for real samples is controlled by the concentration necessary for satisfactory derivatization, not by the capability of the LIF detector. This limitation is true for other reactive moieties, besides isothiocyanate, although some, such as the succinimidyl ester amine-reactive group, may provide better performance [ 10 ]. For the derivatization of proteins using FITC, the true LOD is about the same as for absorbance detection. 2.2. Production of multiple £uorescent derivatives
The derivatization of a protein with a probe results in a distribution of £uorescent derivatives which differ in the number and spatial position of £uorescein labels. This distribution tends to prevent quanti¢cation of proteins by capillary zone electrophoresis due to the partial separation of the distribution. Simply derivatizing the native protein with a large excess of probe in the hopes of forcing the production of a unique derivative with all possible derivatization sites labeled tends not to work. This is largely due to the complexity of the structure of the polypeptide where neighboring residues can sterically hinder complete derivatization or alter the reactivity of the primary amine involved. The derivatization of myoglobin with FITC can be used to demonstrate how this problem originates. Myoglobin is a relatively small protein with a molecular weight of about 17.5 kDa. It possesses 1 N-terminus and 19 lysine residues and so there are 20 different possible derivatization sites. At the pH used for deri-
vatization ( 9.0^9.5 ), the lysine O-amine groups are largely positively charged. Since covalently bound £uorescein will exist as a dianion at this pH [ 11 ], a single positive charge from the O-amine group is exchanged for two negative charges with each £uorescein label attached to the protein. The molecular weight of myoglobin is not changed appreciably by derivatization of the relatively small probe which is only 2% of its native weight. Since capillary zone electrophoresis ( CZE ) separates on the basis of charge to size ratio, the different £uorescent derivatives will possess different migration times. The more £uorescent labels incorporated, the longer the migration time. This is apparent in Fig. 2. Peak identi¢cation in this electropherogram has been previously explained [ 12 ]. Other probes are either similarly negatively charged or neutral, thus similar problems with multiple derivatives will exist. Any successful methodology that uses £uorescent derivatization for low concentration protein analysis must circumvent these two problems brought about by chemical derivatization. We review here a number of avenues various research groups have explored to circumvent these problems. For a more comprehensive review, which deals with the derivatization of both peptides and proteins, for CE and HPLC separations, the reader is directed to the recent publication from Krull et al. [ 13 ].
3. Solutions to problems in the £uorescent derivatization of proteins 3.1. Fluorogenic hydrophobic probes
Swalie and Sepaniak have investigated the use of the £uorescent hydrophobic probes 1-anilinonaphthalene-8-sulfonate ( ANS ) and 2-p-toluidinonaphthalene-6-sulfonate (TNS ) to label proteins [ 6 ]. These probes intercalate into the hydrophobic regions of proteins whereupon their £uorescent quantum yield increases substantially. This allows their use as oncolumn derivatization reagents added to separation buffers as they are non-£uorescent until incorporated into the protein. Although both ANS and TNS are negatively charged at the pH of the electrophoresis buffer used, it was thought that the dynamic nature of the probe-protein binding process avoided the multiple £uorescent derivative problem. The improvement in LOD with this method relative to on-column UV absorbance detection was only modest, however, as protein concentrations below 1037 M could not be
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Fig. 2. A: Electropherogram of native myoglobin. Electrophoretic run buffer: 0.1 M borate, pH 9.28, absorbance detection at 200 nm, effective capillary length: 45 cm. B: Electrophoretic separation of a derivatization reaction between FITC and myoglobin ( 1:1 molar ratio ). Derivatization and electrophoretic run buffer: 0.1 M borate, pH 9.28, £uorescence detection (V ex : 488 nm; Vem : V520 nm ), effective capillary length: 30 cm. Myo-1F refers to FTC-myoglobin with one £uorescein label covalently bound; Myo-2F contains two £uorescein labels covalently bound; etc.
detected. This LOD can now be achieved using UV absorbance detection with extended path length detection cells so the method appears to possess little practical utility. 3.2. Fluorogenic amine-reactive probes and post-column derivatization
Not all amine-reactive probes have such high concentration requirements as FITC for effective derivatization. If the isothiocyanate reactive moiety is changed to a succinimidyl ester, nanomolar concentrations of analyte can be derivatized and detected by CE [ 10 ]. The £uorogenic probes o-phthalaldehyde (OPA ) and naphthalene-2,3-dicarboxaldehyde (NDA ) have similar capabilities [ 14,15 ]. The multiple £uorescent derivatives produced from protein derivatization will still possess different electrophoretic mobilities, however, since the net charge of the derivatives will differ.
Yet OPA and NDA have other attributes which can make them favorable for protein separations with LIF detection. The fact that they are not inherently £uorescent and that the kinetics of the derivatization are extremely rapid allows their use in the post-column derivatization of analyte. Jorgenson's group developed a post-column reactor in the late 1980s ( see Fig. 3 ) which used a reactor tee to join a separation capillary, a labeling reagent capillary and a reaction capillary which also served as the detection cell [ 16 ]. The post-column reactor required sub-second reaction times between the £uorogenic reactant and analyte for appropriate sensitivity. Using OPA, their detection limit for myoglobin was about 1038 M. This represents a 2 order of magnitude improvement in LOD relative to UV absorbance detection. Yeung's group has recently developed another post-column reactor based on a co-axial design which reports similar LOD for proteins [ 17 ].
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Fig. 3. Post-column reactor for the derivatization of proteins with OPA. The separation capillary outlet end was etched with hydro£uoric acid, then inserted into the reaction capillary. OPA in buffer is pumped into the reaction capillary through the annular space between it and the separation capillary where derivatization of eluting proteins can occur followed by their detection. Adapted from Ref. [ 18 ].
No post-column reactor is commercially available at this time, so instrumentation must be built in-house. A number of designs are described in Krull's review [ 18 ]. While reactor construction may not pose much of a problem for some Research and Development laboratories, typical end-users of CE instrumentation may not want to construct their own reactor, particularly if validation is an issue. 3.3. Immunoassays using £uorescent probes
Kennedy's group was the ¢rst to demonstrate the use of competitive immunoassays to circumvent probe concentration requirements [ 19 ]. They developed an assay for insulin by using a commercial preparation of FTC-insulin as a standard reagent, along with a limited amount of monoclonal antibody fragment ( Fab ) to insulin. The assay works by setting up a competition between the standard FTC-insulin and analyte insulin for a limited amount of Fab. CZE is used to separate rapidly the Fab^FTC-insulin complex from free FTCinsulin. The ratio of complex to free FTC-insulin allowed the quanti¢cation of nanomolar concentrations of insulin. Insulin is one of the smallest proteins and contains only three primary amines, two N-termini and a lysine residue. Even so, the researchers were faced with the
multiple derivative problem with the possibility of seven discernible derivatives. The electropherograms of the assay contained three recognizable FTC-insulin derivatives, as demonstrated in Fig. 4, which complicated the procedure. In later work using the same methodology, they isolated a derivative with two £uorescein tags by HPLC to yield single peaks for excess reagent and complex [ 20 ]. HPLC isolation of a unique derivative is possible for small proteins with few primary amines, but for larger proteins with tens of lysine residues, the procedure would likely be unsuccessful. Although £uorescent labeling of proteins produces multiple products, separations can be tuned to separate the broad £uorescent reagent peak from its complex. This was demonstrated in a chip-based separation of FTC-BSA from the immune complex to quantify a monoclonal antibody to BSA in ascites £uid [ 21 ]. In another application, FTC-protein G was used as a labeling reagent which binds to the Fc portion of human IgG ( h-IgG ) [ 22 ]. The methodology was used for the quanti¢cation of h-IgG in serum. In each of these cases the desired separation was performed in one minute or less, so that separation of multiple £uorescent products was minimized, yet satisfactory separation of reagent from complex was achieved. Furthermore, the rapid separation
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Fig. 4. Top electropherogram: 100 nM FTC-insulin. Bottom electropherogram: 100 nM FTC-insulin and 50 nM Fab. Peaks 2, 3 and 5 are FTC-insulin; peaks 1 and 4 are due to the formation of a complex of Fab with FTCinsulin with peaks 2 and 5, respectively. From Ref. [ 21 ], with permission.
ensured that the immune complex had no time to dissociate. Karger's group has developed a non-competitive immunoassay where a F( abP ) antibody fragment was labeled with a £uorophore and used to quantify human growth hormone ( hGH ) [ 23 ]. In this application, the F( abP ) was modi¢ed before derivatization to expose a single thiol group through which selective derivatization could proceed with an iodoacetamide derivative of tetramethylrhodamine. Using this methodology, outlined in Fig. 5, the authors were able to isolate a F( abP ) fragment with a single £uorescent label by preparative isoelectric focusing. This reagent was then used to quantify hGH to pM levels in standard assays using capillary isoelectric focusing; although for serum samples, the limit of detection for hGH was elevated by two orders of magnitude due to background £uorescence. Schmalzing and Nashabeh have recently reviewed CE immunoassays [ 24 ]. They point out that non-competitive immunoassays, where £uorescent antibodies are used in excess, should provide the best sensitivity; but problems ensue due to the dif¢culty in separating the £uorescent immune reagent from the complex. The reasons behind this are two-fold. First, it appears that in many cases, complex formation does not cause a signi¢cant change in electrophoretic mobility relative to the free £uorescent immune reagent. Second, some proteins themselves tend to be somewhat heterogene-
Fig. 5. Production of a single £uorescent label on a F( abP ) fragment. 1: Murine monoclonal antibody is cleaved with pepsin to yield a F( abP )2 fragment; 2: F( abP )2 fragment is reduced with mercaptoethylamine, to produce a F( abP ) fragment with three exposed thiols; 3: two of the thiol groups are oxidized using copper ( II ) sulfate to form a disul¢de bridge resulting in a single thiol F( abP ) fragment; 4: the single thiol F( abP ) fragment is derivatized with tetramethylrhodamine-5,6-iodoacetamide. Adapted from [ 23 ].
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Fig. 6. Separation of FQ-labeled bovine serum albumin ( BSA ), L-lactoglobulin A and K-lactalbumin labeled at a concentration of 5, 10 and 15 nM, respectively. From [ 24 ], with permission.
ous due to various post-translational modi¢cations. This is certainly true of IgG antibodies. Thus, the identi¢cation and quanti¢cation of protein analyte based on complex peak height measurements can be problematic and the problems tend to be compounded at lower analyte concentrations. 3.4. Uncharged amine-reactive £uorogenic probes and sub-micellar anionic surfactantcontaining separation buffers
Dovichi's group has recently developed a method for the pM assay of native proteins using the £uorogenic amine-reactive probe 5-furoylquinoline-3-carboxaldehyde ( FQ ) and sub-micellar concentrations of the anionic surfactant sodium dodecyl sulfate (SDS ) added to the separation buffer [ 25 ]. FQ uses similar chemistry to OPA and NDA thus possesses the ability to derivatize low concentration analyte. The sub-micellar concentrations of SDS are used essentially as a charge to mass balance for FQ-derivatized proteins. It is thought that SDS, present in sub-micellar
concentrations, binds to proteins through ion pairing, thus the anionic head group will interact with positively charged lysine residues. This ion pairing essentially converts lysine into an uncharged residue with an increase in mass of 265 amu. When derivatized with FQ, a lysine residue is converted into an uncharged isoindole with an increase in mass of 283 amu. Presumably all surface lysine residues either ion pair or are converted into isoindoles, thus the multiple £uorescent derivatives all possess similar electrophoretic mobilities and thus migration times. Separation ef¢ciencies typical of native proteins can be achieved and the LOD for a number of proteins was demonstrated to be in the range of 0.01^1 nM. This recently developed method has much potential for practical £uorescence detection of proteins separated by CE. There is an area of concern with this method, however. The CZE separation of three model proteins demonstrated in the manuscript ( see Fig. 6 ) was not baseline resolved which suggests that selectivity may be somewhat compromised by the addition of submicellar concentrations of surfactant
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Fig. 7. Separation of FTC-proteins in a denaturing 4% T linear polyacrylamide gel with LIF detection. Peak identi¢cations are as follows: 1: insulin; 2: cytochrome c; 3: lysozyme; 4: myoglobin; 5: soybean trypsin inhibitor; 6: chymotrypsinogen; 7: carbonic anhydrase; 8: bovine serum albumin; 9: phosphorylase b. From [ 27 ], with permission.
to the electrophoresis run buffer. This would be due presumably to all the positive charge associated with the protein, from both arginine and lysine residues, being extinguished by ion pairing with dodecyl sulfate. This degree of positive charge extinguishment will not be present from conventional £uorescence labeling. 3.5. Sodium dodecyl sulfate^capillary gel electrophoresis (SDS-CGE )
A number of groups have investigated £uorescent derivatization of proteins for size-based separations using SDS-CGE. Since most proteins bind a constant amount of SDS based on a mass ratio of 1.4 g of SDS per g of protein, the charge alteration on lysine residues brought about by £uorescent derivatization is overwhelmed. Thus the multiple £uorescent derivatives all possess similar electrophoretic mobilities. To emphasize this principle, consider the protein bovine serum albumin ( BSA ), one of the most commonly used marker proteins in slab gel separations. BSA has a molecular weight of about 66 kDa, compared to 288 amu for SDS, thus there should be about 320 dodecyl sulfate molecules, each contributing a
negative charge, binding to each BSA molecule during an SDS-CGE separation. If an FTC-BSA distribution exists with between 1 and 10 £uorescein molecules bound to the BSA population, the range of charge difference in the population will be 18 since a 32 charge is introduced into the protein for each £uorescein label. It is evident that the 320 negative charges from SDS binding will overwhelm this range of charge difference. There will be some broadening, but the FTC-BSA distribution will migrate as a unique zone. Gump and Monnig demonstrated this principle using OPA- and NDA-derivatized proteins [ 26 ], Hogan's group used other amine-reactive £uorogenic probes, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl ) and 4-£uoro-7-nitrobenzofurazan (NBDF ) [ 27 ] and Beale's group demonstrated that FTCproteins could also be separated by SDS-CGE without multiple derivative problems ( see Fig. 7 ) [ 28 ]. In each of these cases, however, WM or higher native protein concentrations were derivatized. Dovichi's group has demonstrated the use of FQ-labeling for LIF detection in SDS-CGE with the ability to detect sub-1037 M concentrations of protein [ 25 ]. Our laboratory has recently demonstrated the ability to achieve
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Fig. 8. Transglutaminase-mediated tetramethylrhodamine^cadaverine ( A ) derivatization of a glutamine residue in a protein ( B ) to yield a tetramethylrhodamine^protein derivative ( C ).
an LOD in SDS-CGE which is similar to silver staining in slab gel electrophoresis [ 29 ]. We use the noncovalent, £uorogenic probe Sypro Red1 to label the SDS^protein complex. Using this technique, we have labeled 1.5 nM concentrations of BSA and demonstrated a calculated LOD of 73 pM. Although these methods are suitable for size-based separations, the need for boiling the sample and the use of a large excess of SDS ensures that the proteins are denatured. Thus this technique cannot be considered a general panacea for low concentration protein analysis since the separation of native proteins is not possible. This can be problematic for complex samples as the separation ef¢ciency in SDS-CGE, especially when the proteins exist as distributions of derivatives, tends to be poor relative to CZE. Furthermore, there is a lack of data providing peak height or peak area reproducibility for £uorescently derivatized proteins
separated by SDS-CGE. Without adequate reproducibility, quantitative protein separations are not possible. 3.6. Solid phase labeling
Solid phase labeling is an interesting strategy to circumvent probe concentration requirements. Dovichi's group have used an Immobilon CD membrane to immobilize insulin chain B ( Ins B ) in an in-house constructed solid phase reactor [ 30 ]. Ins B can then be labeled with vast excess amounts of £uorescent probe, in this case FQ, without interference from £uorescent products due to hydrolysis and secondary reactions. These unwanted products are £ushed away before extraction of Ins B with low pH buffer and off-line analysis by micellar electrokinetic chromatography. This system was able to derivatize 1038 M Ins B
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by using a preconcentration step in the solid phase reactor. While this technique works for Ins B with only two possible derivatization sites, any protein labeled by this technique will yield multiple £uorescent derivatives. Krull's group has developed a number of methodologies for the derivatization of proteins for analysis by HPLC. These methodologies rely on solid phase labeling where the £uorescent derivatizing reagent, 6-aminoquinoline carbamate ( 6-AQC ), is immobilized onto a styrene^divinylbenzene support. The immobilized £uorophore possesses an activated linkage that will allow the nucleophilic addition of the 6AQC to a protein as it £ows past [ 31 ]. Using a speci¢c type of support with well-controlled physical properties, they have performed size selective derivatizations of proteins with selective labeling of only those primary amine( s ) that can access the interior of the support pores [ 32 ]. These methodologies may be of practical use for CE separations also, as the authors suggest, although it may prove that the signi¢cantly improved separation ef¢ciency of CE may partially resolve a distribution of products that HPLC can not.
4. Summary Although £uorescent derivatization of proteins for capillary electrophoretic separations is problematic, a number of useful methodologies have been described in this review. Each has its advantages and disadvantages, which suggests that there is room for improvement. A number of research groups are examining new ways of performing £uorescent derivatization for practical detection of proteins by CE. Krull's group is currently investigating the use of excess amounts of £uorescent probe in derivatization reactions in order to force complete derivatization [ 33 ]. The stoichiometric excess of probe must be relative to the number of primary amines in the analyte protein. In addition, it appears that the protein must be denatured to expose all groups to the probe. As one would expect, this technique is protein dependent in that some analytes require different degrees of denaturation to expose all N-termini and lysine residues to possible derivatization. Excellent results have been achieved for insulin where MALDI^TOF experiments have con¢rmed the derivatization of both N-termini and the lone lysine residue. The CZE separation of the derivatization reaction mixture demonstrates a unique insulin derivative and probe hydrolysis products. Furthermore, the sep-
aration ef¢ciency of the derivative is improved signi¢cantly relative to the native form [ 33 ]. The complete derivatization of other proteins is currently being examined. Although low concentration derivatization has not so far been investigated, the succinimidyl ester amine-reactive moiety used in this study has the ability to derivatize amino acids at nanomolar concentrations [ 10 ], thus the capability to derivative low protein concentrations could follow. Our group is currently investigating the use of the enzyme transglutaminase for the labeling of glutamine residues in proteins with a £uorescent, uncharged derivative of cadaverine ( for example: tetramethylrhodamine^cadaverine ). In this method ( Fig. 8 ), an uncharged residue is derivatized with an uncharged £uorophore which will provide a distribution of derivatives with the same charge and thus similar electrophoretic mobilities. Furthermore, the need for the enzyme should narrow the distribution of products generated in the derivatization reaction since presumably the protein to be derivatized must access the active site of the enzyme. One can anticipate steric hindrances limiting the number of glutamine residues that can be derivatized. If the probe concentration requirements are high with this method, the derivative could be used as an immune reagent for the competitive immunoassay of the native protein, as described by Kennedy's group.
5. Abbreviations ANS 6-AQC BSA CE CZE FITC FQ FTC hGH HPLC IgG Ins B Vex LIF LOD NBD-Cl NBD-F NDA OPA SDS SDS-CGE TNS
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1-anilinonaphthalene-8-sulfonate 6-aminoquinoline carbamate bovine serum albumin capillary electrophoresis capillary zone electrophoresis £uorescein isothiocyanate 5-furoylquinoline-3-carboxaldehyde £uorescein thiocarbamyl human growth hormone high performance liquid chromatography immunoglobulin G insulin, chain B wavelength of maximum excitation laser-induced £uorescence limit of detection 4-nitrobenz-2-oxa-1,3-diazole 4-£uoro-7-nitrobenzofurazan naphthalene-2,3-dicarboxaldehyde o-phthalaldehyde sodium dodecyl sulfate sodium dodecyl sulfate^capillary gel electrophoresis 2-p-toluidinonaphthalene-6-sulfonate
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References [ 1 ] Various authors, in J.P. Landers ( Editor ), The CRC Handbook of Capillary Electrophoresis, 2nd Edition, CRC Press, Boca Raton, FL, 1997. [ 2 ] A. Kunkel, M. Degenhardt, B. Schirm, H. Waëtzig, J. Chromatogr. A 768 ( 1997 ) 17. [ 3 ] D.N. Heiger, High Performance Capillary Electrophoresis ^ An Introduction, Hewlett-Packard Co. Publication Number 12-5091-6199E, p. 100. [ 4 ] S.E. Moring, R.T. Reel, R.E.J. van Soest, Anal. Chem. 65 ( 1993 ) 3454. [ 5 ] HP3D CE High Sensitivity Cell, Hewlett-Packard Co. Publication Number 12-5965-5984E. [ 6 ] D.F. Swaile, M.J. Sepaniak, J. Liq. Chromatogr. 14 ( 1991 ) 869. [ 7 ] T.T. Lee, E.S. Yeung, J. Chromatogr. 595 ( 1992 ) 319. [ 8 ] D.M. Paquette, R. Sing, P.R. Banks and K.C. Waldron, J. Chromatogr. B, ( 1998 ) in press. [ 9 ] Y.-F. Cheng, N.J. Dovichi, Science 242 ( 1988 ) 562. [ 10 ] S.K. Lau, F. Zaccardo, M. Little, P. Banks, J. Chromatogr. A 809 ( 1998 ) 203. [ 11 ] N. Klonis, W.H. Sawyer, J. Fluoresc. 6 ( 1996 ) 147. [ 12 ] P.R. Banks, D.M. Paquette, J. Chromatogr. A 693 ( 1995 ) 145. [ 13 ] I.S. Krull, R. Strong, Z. Sosic, B.-Y. Cho, S.C. Beale, C.-C. Wang, S. Cohen, J. Chromatogr. B 699 ( 1997 ) 173. [ 14 ] O. Orwar, M. Sandberg, I. Jacobson, M. Sundahl, S. Folestad, Anal. Chem. 67 ( 1995 ) 4261. [ 15 ] T. Ueda, R. Mitchell, F. Kitamura, T. Metcalf, T. Kuwana, A. Nakamoto, J. Chromatogr. 593 ( 1992 ) 265. [ 16 ] B. Nickerson, J.W. Jorgenson, J. Chromatogr. 480 ( 1989 ) 157. [ 17 ] L. Zhang, E.S. Yeung, J. Chromatogr. A 734 ( 1996 ) 331. [ 18 ] M.E. Szulc, I.S. Krull, J. Chromatogr. A 659 ( 1994 ) 231. [ 19 ] N.M. Schultz, R.T. Kennedy, Anal. Chem. 65 ( 1993 ) 3161. [ 20 ] N.M. Schultz, L. Huang, R.T. Kennedy, Anal. Chem. 67 ( 1995 ) 924. [ 21 ] N. Chiem, D.J. Harrison, Anal. Chem. 69 ( 1997 ) 373. [ 22 ] O.W. Reif, R. Lausch, T. Scheper, R. Freitag, Anal. Chem. 66 ( 1994 ) 4027.
[ 23 ] K. Shimura, B.L. Karger, Anal. Chem. 66 ( 1994 ) 9. [ 24 ] D. Schmalzing, W. Nashabeh, Electrophoresis 18 ( 1997 ) 2184. [ 25 ] D.M. Pinto, E.A. Arriaga, D. Craig, J. Angelova, N. Sharma, H. Ahmadzadeh, C.A. Boulet, N.J. Dovichi, Anal. Chem. 69 ( 1997 ) 3015. [ 26 ] E.L. Gump, C.A. Monnig, J. Chromatogr. A 715 ( 1995 ) 167. [ 27 ] E.T. Wise, N. Singh, B.L. Hogan, J. Chromatogr. A 746 ( 1996 ) 109. [ 28 ] C.-C. Wang, S.C. Beale, J. Chromatogr. A 756 ( 1996 ) 245. [ 29 ] M.D. Harvey, D. Bandilla and P.R. Banks, Electrophoresis, ( 1998 ) in press. [ 30 ] D.M. Pinto, E.A. Arriaga, S. Sia, Z. Li, N.J. Dovichi, Electrophoresis 16 ( 1995 ) 534. [ 31 ] G. Li, J. Yu, I.S. Krull, S. Cohen, J. Liq. Chromatogr. 18 ( 1995 ) 3889. [ 32 ] M.E. Szulc, P. Swett, I.S. Krull, Biomed. Chromatogr. 11 ( 1997 ) 207. [ 33 ] B.-Y. Cho, H. Liu and I.S. Krull, unpublished results. Peter R. Banks was born a British citizen in Bombay (Mumbai ), India in 1959. In 1961, he emigrated to Canada with his parents and became a Canadian citizen in 1965. He received a BSc in Chemistry from the University of British Columbia in 1986 and a PhD in Analytical Chemistry from the same institution in 1992 under the guidance of Michael W. Blades. His postdoctoral studies in the laboratories of Norman J. Dovichi at the University of Alberta were funded by an NSERC Postdoctoral Fellowship awarded for 1992^1994. Dr. Banks is currently an Assistant Professor in the Department of Chemistry and Biochemistry at Concordia University where he has been since 1994. His main research interests are directed towards developing methodology for the low concentration analysis of proteins, especially in clinical samples; the analysis of neurotransmitters from microdialysate samples using £uorescent derivatization; and the use of non-aqueous capillary electrophoresis for the analysis of non-polar analyte.
TrAC / Internet column In order to inform analytical chemists about the Internet and the role it could play in their lives, Dr. Michael Guilhaus was invited to become a Contributing Editor of TrAC. The Internet Column has now become a feature of the journal. The column can also be found on the World Wide Web. Anyone interested in contributing to this column is invited to contact Michael Guilhaus at: Mike^[email protected] The Internet Column articles of TrAC can also be found on the Web. If you have a browser, to access the TrAC column on the Web simply point to: http: / www.elsevier.nl / locate / trac
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mauro, O. Zerbinati, M.L. Tosato, Environ. Sci. Technol. 24 ( 1990 ) 1559. [ 28 ] M. Bideau, B. Claudel, L. Faure, M. Rachimoellah, J. Photochem. 39 ( 1987 ) 107. [ 29 ] J.J. Pignatello, Environ. Sci. Technol. 26 ( 1992 ) 944. [ 30 ] M. Barbeni, C. Minero, E. Pelizzetti, E. Borgarello, N. Serpone, Chemosphere 16 ( 1987 ) 2225. [ 31 ] D.L. Sedlak, A.W. Andren, Environ. Sci. Technol. 25 ( 1991 ) 777. [ 32 ] C. Maillard, Ch. Guillard, P. Pichat, Chemosphere 24 ( 1992 ) 1085. [ 33 ] M. Halmann, J. Photochem. Photobiol. A Chem. 66 ( 1992 ) 215. [ 34 ] A. Mills, R. Davies and D. Worsley, Chem. Soc. Rev., ( 1993 ) 417.
[ 35 ] C.K. Graëtzel, M. Jitousek, M. Graëtzel, J. Mol. Catal. 60 ( 1990 ) 375. [ 36 ] C.S. Turchi, D.F. Ollis, J. Catal. 122 ( 1990 ) 178. [ 37 ] T. Hisanaga, K. Harada, K. Tanaka, J. Photochem. Photobiol. A Chem. 54 ( 1990 ) 113. [ 38 ] E. Pramauro, M. Vincentini, V. Augugliaro, L. Palmisano, Environ. Sci. Technol. 27 ( 1993 ) 1790. [ 39 ] G. Penìuela and D. Barceloè, J. Chromatogr. A 823 ( 1998 ) 81. [ 40 ] C.H. Walling, Acc. Chem. Res. 8 ( 1975 ) 125. [ 41 ] W.G. Barb, J.H. Baxendale, P. George, K.R. Hargrave, Trans. Faraday Soc. 47 ( 1951 ) 591. [ 42 ] G.K. Pandit, S. Pal, A.K. Das, J. Agric. Food Chem. 43 ( 1995 ) 171.
Fluorescent derivatization for low concentration protein analysis by capillary electrophoresis Peter R. Banks*
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Blvd. W., Montreal, Que. H3G 1M8, Canada Fluorescence derivatization can allow for the low concentration analysis of proteins by capillary electrophoresis. Major problems arising from inef¢cient chemistry and multiple derivatives must be overcome, however, for the method to be successful. A number of methods are discussed in this review. z1998 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence; Derivatization; Protein; Capillary electrophoresis
1. Introduction Although capillary electrophoresis ( CE ) is a relatively new separation technique when compared to more established methods such as high performance liquid chromatography ( HPLC ), gas chromatography and slab gel electrophoresis, its numerous advantages *Fax: +1 (514) 848-2868. E-mail: [email protected]
have resulted in many diverse research efforts and widespread use. These advantages include high separation ef¢ciency, analysis speed, instrumentation simplicity and the ability to perform unique analyses, such as the examination of the contents of a single cell [ 1 ]. Furthermore, inferior analytical performance due to both poor migration time and injection reproducibility has been overcome by the advent of new commercial instrumentation where reproducibilities below 1% RSD are now standard [ 2 ]. Despite these advantages and advances, detection is still a problem for analyses requiring the detection of low concentration analyte. The primary focus of my laboratory is on low concentration protein analysis by capillary electrophoresis, thus detection is one of the main problems I experience in my research efforts. The ubiquitous deuterium lamp-based UV absorbance detector is unsuited for low concentration protein analysis since the short path length ( approx. 39 Wm for a 50 Wm I.D. capillary ) of the detection cell limits the determination of proteins to concentrations typically greater than micromolar (WM ) levels. Even with extended path length devices such as a bubble cell [ 3 ], Z-cell [ 4 ] or high sensitivity cell [ 5 ], limits of detection ( LOD )
0165-9936/98/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 0 7 3 - 9
ß 1998 Elsevier Science B.V. All rights reserved.
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Fig. 1. Derivatization of a lysine residue in a protein with FITC to produce a £uorescein thiocarbamyl derivative.
are only extended by one order of magnitude, at best. Thus I ( and others ) investigate alternative detection methods which can provide lower LOD. An obvious candidate for low concentration detection is £uorescence, especially when the excitation source is a laser. Proteins are intrinsic £uorophores if they contain aromatic amino acid residues. Both excitation and emission are in the UV, however, which limits the type of laser that can be used. Swaile and Sepaniak have used a frequency doubled Ar laser ( 257 nm ) [ 6 ], Lee and Yeung used the 275.4 nm line from a similar laser [ 7 ], and a number of groups, including mine [ 8 ], have used a KrF excimer laser ( 248 nm ) for the detection of proteins separated by CE. Systems using these lasers can generally provide nanomolar ( nM ) LOD for proteins. Unfortunately, these UV lasers tend to be expensive compared to lasers that operate in the visible. The Ar lasers used for native protein detection must be water-cooled to generate mW UV power necessary for optimal £uorescence generation. These lasers are about an order of magnitude more expensive than their air-cooled cousins which produce mW power levels in the visible ( 488 nm ). Even the KrF laser is about three times more expensive than this laser.
For this reason, most laser-induced £uorescence ( LIF ) detectors for CE separations use inexpensive lasers that operate in the visible or near UV. The Ar laser operating at 488 nm is the most common, but other lasers used include the UV line of the He Cd laser ( 325 nm ), the green He Ne laser ( 543.5 nm ) and increasingly, very inexpensive diode lasers operating in the red ( i.e. 635 nm ). LIF detection with visible lasers is also available in commercial instrumentation: for example, Beckman instruments offers excitation at both 488 and 635 nm in their P / ACE systems. Few native proteins contain a £uorophore which can be excited by these lasers. Yet proteins can be converted into suitable £uorescent derivatives by chemical derivatization, typically using amine-reactive £uorescent or £uorogenic probes. These probes contain an amine-reactive moiety or are converted into a £uorophore by their covalent attachment to proteins through primary amines, such as the N-terminus or lysine residues. This process is demonstrated in Fig. 1 for the derivatization of a lysine residue in a protein with the most commonly used £uorescent derivatizing reagent, £uorescein isothiocyanate ( FITC ). Using this probe, picomolar ( pM ) concentrations of £uorescein
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thiocarbamyl ( FTC )-amino acids have been detected [ 9 ].
2. Problems in the £uorescent derivatization of proteins 2.1. Probe concentration requirements
In order to detect picomolar concentrations of FTCamino acids, they must ¢rst be derivatized at high concentration ( typically 1034 M ) then diluted by a factor of 108 . The high amino acid concentration for the derivatization reaction is required because the chemistry of FITC derivatization of primary amines is slow and inef¢cient. My group has demonstrated that the lowest concentration of amino acid that can be recognizably derivatized is about 1036 M [ 10 ]. This limitation is also apparent for protein derivatization. Thus the LOD for real samples is controlled by the concentration necessary for satisfactory derivatization, not by the capability of the LIF detector. This limitation is true for other reactive moieties, besides isothiocyanate, although some, such as the succinimidyl ester amine-reactive group, may provide better performance [ 10 ]. For the derivatization of proteins using FITC, the true LOD is about the same as for absorbance detection. 2.2. Production of multiple £uorescent derivatives
The derivatization of a protein with a probe results in a distribution of £uorescent derivatives which differ in the number and spatial position of £uorescein labels. This distribution tends to prevent quanti¢cation of proteins by capillary zone electrophoresis due to the partial separation of the distribution. Simply derivatizing the native protein with a large excess of probe in the hopes of forcing the production of a unique derivative with all possible derivatization sites labeled tends not to work. This is largely due to the complexity of the structure of the polypeptide where neighboring residues can sterically hinder complete derivatization or alter the reactivity of the primary amine involved. The derivatization of myoglobin with FITC can be used to demonstrate how this problem originates. Myoglobin is a relatively small protein with a molecular weight of about 17.5 kDa. It possesses 1 N-terminus and 19 lysine residues and so there are 20 different possible derivatization sites. At the pH used for deri-
vatization ( 9.0^9.5 ), the lysine O-amine groups are largely positively charged. Since covalently bound £uorescein will exist as a dianion at this pH [ 11 ], a single positive charge from the O-amine group is exchanged for two negative charges with each £uorescein label attached to the protein. The molecular weight of myoglobin is not changed appreciably by derivatization of the relatively small probe which is only 2% of its native weight. Since capillary zone electrophoresis ( CZE ) separates on the basis of charge to size ratio, the different £uorescent derivatives will possess different migration times. The more £uorescent labels incorporated, the longer the migration time. This is apparent in Fig. 2. Peak identi¢cation in this electropherogram has been previously explained [ 12 ]. Other probes are either similarly negatively charged or neutral, thus similar problems with multiple derivatives will exist. Any successful methodology that uses £uorescent derivatization for low concentration protein analysis must circumvent these two problems brought about by chemical derivatization. We review here a number of avenues various research groups have explored to circumvent these problems. For a more comprehensive review, which deals with the derivatization of both peptides and proteins, for CE and HPLC separations, the reader is directed to the recent publication from Krull et al. [ 13 ].
3. Solutions to problems in the £uorescent derivatization of proteins 3.1. Fluorogenic hydrophobic probes
Swalie and Sepaniak have investigated the use of the £uorescent hydrophobic probes 1-anilinonaphthalene-8-sulfonate ( ANS ) and 2-p-toluidinonaphthalene-6-sulfonate (TNS ) to label proteins [ 6 ]. These probes intercalate into the hydrophobic regions of proteins whereupon their £uorescent quantum yield increases substantially. This allows their use as oncolumn derivatization reagents added to separation buffers as they are non-£uorescent until incorporated into the protein. Although both ANS and TNS are negatively charged at the pH of the electrophoresis buffer used, it was thought that the dynamic nature of the probe-protein binding process avoided the multiple £uorescent derivative problem. The improvement in LOD with this method relative to on-column UV absorbance detection was only modest, however, as protein concentrations below 1037 M could not be
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Fig. 2. A: Electropherogram of native myoglobin. Electrophoretic run buffer: 0.1 M borate, pH 9.28, absorbance detection at 200 nm, effective capillary length: 45 cm. B: Electrophoretic separation of a derivatization reaction between FITC and myoglobin ( 1:1 molar ratio ). Derivatization and electrophoretic run buffer: 0.1 M borate, pH 9.28, £uorescence detection (V ex : 488 nm; Vem : V520 nm ), effective capillary length: 30 cm. Myo-1F refers to FTC-myoglobin with one £uorescein label covalently bound; Myo-2F contains two £uorescein labels covalently bound; etc.
detected. This LOD can now be achieved using UV absorbance detection with extended path length detection cells so the method appears to possess little practical utility. 3.2. Fluorogenic amine-reactive probes and post-column derivatization
Not all amine-reactive probes have such high concentration requirements as FITC for effective derivatization. If the isothiocyanate reactive moiety is changed to a succinimidyl ester, nanomolar concentrations of analyte can be derivatized and detected by CE [ 10 ]. The £uorogenic probes o-phthalaldehyde (OPA ) and naphthalene-2,3-dicarboxaldehyde (NDA ) have similar capabilities [ 14,15 ]. The multiple £uorescent derivatives produced from protein derivatization will still possess different electrophoretic mobilities, however, since the net charge of the derivatives will differ.
Yet OPA and NDA have other attributes which can make them favorable for protein separations with LIF detection. The fact that they are not inherently £uorescent and that the kinetics of the derivatization are extremely rapid allows their use in the post-column derivatization of analyte. Jorgenson's group developed a post-column reactor in the late 1980s ( see Fig. 3 ) which used a reactor tee to join a separation capillary, a labeling reagent capillary and a reaction capillary which also served as the detection cell [ 16 ]. The post-column reactor required sub-second reaction times between the £uorogenic reactant and analyte for appropriate sensitivity. Using OPA, their detection limit for myoglobin was about 1038 M. This represents a 2 order of magnitude improvement in LOD relative to UV absorbance detection. Yeung's group has recently developed another post-column reactor based on a co-axial design which reports similar LOD for proteins [ 17 ].
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Fig. 3. Post-column reactor for the derivatization of proteins with OPA. The separation capillary outlet end was etched with hydro£uoric acid, then inserted into the reaction capillary. OPA in buffer is pumped into the reaction capillary through the annular space between it and the separation capillary where derivatization of eluting proteins can occur followed by their detection. Adapted from Ref. [ 18 ].
No post-column reactor is commercially available at this time, so instrumentation must be built in-house. A number of designs are described in Krull's review [ 18 ]. While reactor construction may not pose much of a problem for some Research and Development laboratories, typical end-users of CE instrumentation may not want to construct their own reactor, particularly if validation is an issue. 3.3. Immunoassays using £uorescent probes
Kennedy's group was the ¢rst to demonstrate the use of competitive immunoassays to circumvent probe concentration requirements [ 19 ]. They developed an assay for insulin by using a commercial preparation of FTC-insulin as a standard reagent, along with a limited amount of monoclonal antibody fragment ( Fab ) to insulin. The assay works by setting up a competition between the standard FTC-insulin and analyte insulin for a limited amount of Fab. CZE is used to separate rapidly the Fab^FTC-insulin complex from free FTCinsulin. The ratio of complex to free FTC-insulin allowed the quanti¢cation of nanomolar concentrations of insulin. Insulin is one of the smallest proteins and contains only three primary amines, two N-termini and a lysine residue. Even so, the researchers were faced with the
multiple derivative problem with the possibility of seven discernible derivatives. The electropherograms of the assay contained three recognizable FTC-insulin derivatives, as demonstrated in Fig. 4, which complicated the procedure. In later work using the same methodology, they isolated a derivative with two £uorescein tags by HPLC to yield single peaks for excess reagent and complex [ 20 ]. HPLC isolation of a unique derivative is possible for small proteins with few primary amines, but for larger proteins with tens of lysine residues, the procedure would likely be unsuccessful. Although £uorescent labeling of proteins produces multiple products, separations can be tuned to separate the broad £uorescent reagent peak from its complex. This was demonstrated in a chip-based separation of FTC-BSA from the immune complex to quantify a monoclonal antibody to BSA in ascites £uid [ 21 ]. In another application, FTC-protein G was used as a labeling reagent which binds to the Fc portion of human IgG ( h-IgG ) [ 22 ]. The methodology was used for the quanti¢cation of h-IgG in serum. In each of these cases the desired separation was performed in one minute or less, so that separation of multiple £uorescent products was minimized, yet satisfactory separation of reagent from complex was achieved. Furthermore, the rapid separation
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Fig. 4. Top electropherogram: 100 nM FTC-insulin. Bottom electropherogram: 100 nM FTC-insulin and 50 nM Fab. Peaks 2, 3 and 5 are FTC-insulin; peaks 1 and 4 are due to the formation of a complex of Fab with FTCinsulin with peaks 2 and 5, respectively. From Ref. [ 21 ], with permission.
ensured that the immune complex had no time to dissociate. Karger's group has developed a non-competitive immunoassay where a F( abP ) antibody fragment was labeled with a £uorophore and used to quantify human growth hormone ( hGH ) [ 23 ]. In this application, the F( abP ) was modi¢ed before derivatization to expose a single thiol group through which selective derivatization could proceed with an iodoacetamide derivative of tetramethylrhodamine. Using this methodology, outlined in Fig. 5, the authors were able to isolate a F( abP ) fragment with a single £uorescent label by preparative isoelectric focusing. This reagent was then used to quantify hGH to pM levels in standard assays using capillary isoelectric focusing; although for serum samples, the limit of detection for hGH was elevated by two orders of magnitude due to background £uorescence. Schmalzing and Nashabeh have recently reviewed CE immunoassays [ 24 ]. They point out that non-competitive immunoassays, where £uorescent antibodies are used in excess, should provide the best sensitivity; but problems ensue due to the dif¢culty in separating the £uorescent immune reagent from the complex. The reasons behind this are two-fold. First, it appears that in many cases, complex formation does not cause a signi¢cant change in electrophoretic mobility relative to the free £uorescent immune reagent. Second, some proteins themselves tend to be somewhat heterogene-
Fig. 5. Production of a single £uorescent label on a F( abP ) fragment. 1: Murine monoclonal antibody is cleaved with pepsin to yield a F( abP )2 fragment; 2: F( abP )2 fragment is reduced with mercaptoethylamine, to produce a F( abP ) fragment with three exposed thiols; 3: two of the thiol groups are oxidized using copper ( II ) sulfate to form a disul¢de bridge resulting in a single thiol F( abP ) fragment; 4: the single thiol F( abP ) fragment is derivatized with tetramethylrhodamine-5,6-iodoacetamide. Adapted from [ 23 ].
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Fig. 6. Separation of FQ-labeled bovine serum albumin ( BSA ), L-lactoglobulin A and K-lactalbumin labeled at a concentration of 5, 10 and 15 nM, respectively. From [ 24 ], with permission.
ous due to various post-translational modi¢cations. This is certainly true of IgG antibodies. Thus, the identi¢cation and quanti¢cation of protein analyte based on complex peak height measurements can be problematic and the problems tend to be compounded at lower analyte concentrations. 3.4. Uncharged amine-reactive £uorogenic probes and sub-micellar anionic surfactantcontaining separation buffers
Dovichi's group has recently developed a method for the pM assay of native proteins using the £uorogenic amine-reactive probe 5-furoylquinoline-3-carboxaldehyde ( FQ ) and sub-micellar concentrations of the anionic surfactant sodium dodecyl sulfate (SDS ) added to the separation buffer [ 25 ]. FQ uses similar chemistry to OPA and NDA thus possesses the ability to derivatize low concentration analyte. The sub-micellar concentrations of SDS are used essentially as a charge to mass balance for FQ-derivatized proteins. It is thought that SDS, present in sub-micellar
concentrations, binds to proteins through ion pairing, thus the anionic head group will interact with positively charged lysine residues. This ion pairing essentially converts lysine into an uncharged residue with an increase in mass of 265 amu. When derivatized with FQ, a lysine residue is converted into an uncharged isoindole with an increase in mass of 283 amu. Presumably all surface lysine residues either ion pair or are converted into isoindoles, thus the multiple £uorescent derivatives all possess similar electrophoretic mobilities and thus migration times. Separation ef¢ciencies typical of native proteins can be achieved and the LOD for a number of proteins was demonstrated to be in the range of 0.01^1 nM. This recently developed method has much potential for practical £uorescence detection of proteins separated by CE. There is an area of concern with this method, however. The CZE separation of three model proteins demonstrated in the manuscript ( see Fig. 6 ) was not baseline resolved which suggests that selectivity may be somewhat compromised by the addition of submicellar concentrations of surfactant
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Fig. 7. Separation of FTC-proteins in a denaturing 4% T linear polyacrylamide gel with LIF detection. Peak identi¢cations are as follows: 1: insulin; 2: cytochrome c; 3: lysozyme; 4: myoglobin; 5: soybean trypsin inhibitor; 6: chymotrypsinogen; 7: carbonic anhydrase; 8: bovine serum albumin; 9: phosphorylase b. From [ 27 ], with permission.
to the electrophoresis run buffer. This would be due presumably to all the positive charge associated with the protein, from both arginine and lysine residues, being extinguished by ion pairing with dodecyl sulfate. This degree of positive charge extinguishment will not be present from conventional £uorescence labeling. 3.5. Sodium dodecyl sulfate^capillary gel electrophoresis (SDS-CGE )
A number of groups have investigated £uorescent derivatization of proteins for size-based separations using SDS-CGE. Since most proteins bind a constant amount of SDS based on a mass ratio of 1.4 g of SDS per g of protein, the charge alteration on lysine residues brought about by £uorescent derivatization is overwhelmed. Thus the multiple £uorescent derivatives all possess similar electrophoretic mobilities. To emphasize this principle, consider the protein bovine serum albumin ( BSA ), one of the most commonly used marker proteins in slab gel separations. BSA has a molecular weight of about 66 kDa, compared to 288 amu for SDS, thus there should be about 320 dodecyl sulfate molecules, each contributing a
negative charge, binding to each BSA molecule during an SDS-CGE separation. If an FTC-BSA distribution exists with between 1 and 10 £uorescein molecules bound to the BSA population, the range of charge difference in the population will be 18 since a 32 charge is introduced into the protein for each £uorescein label. It is evident that the 320 negative charges from SDS binding will overwhelm this range of charge difference. There will be some broadening, but the FTC-BSA distribution will migrate as a unique zone. Gump and Monnig demonstrated this principle using OPA- and NDA-derivatized proteins [ 26 ], Hogan's group used other amine-reactive £uorogenic probes, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl ) and 4-£uoro-7-nitrobenzofurazan (NBDF ) [ 27 ] and Beale's group demonstrated that FTCproteins could also be separated by SDS-CGE without multiple derivative problems ( see Fig. 7 ) [ 28 ]. In each of these cases, however, WM or higher native protein concentrations were derivatized. Dovichi's group has demonstrated the use of FQ-labeling for LIF detection in SDS-CGE with the ability to detect sub-1037 M concentrations of protein [ 25 ]. Our laboratory has recently demonstrated the ability to achieve
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Fig. 8. Transglutaminase-mediated tetramethylrhodamine^cadaverine ( A ) derivatization of a glutamine residue in a protein ( B ) to yield a tetramethylrhodamine^protein derivative ( C ).
an LOD in SDS-CGE which is similar to silver staining in slab gel electrophoresis [ 29 ]. We use the noncovalent, £uorogenic probe Sypro Red1 to label the SDS^protein complex. Using this technique, we have labeled 1.5 nM concentrations of BSA and demonstrated a calculated LOD of 73 pM. Although these methods are suitable for size-based separations, the need for boiling the sample and the use of a large excess of SDS ensures that the proteins are denatured. Thus this technique cannot be considered a general panacea for low concentration protein analysis since the separation of native proteins is not possible. This can be problematic for complex samples as the separation ef¢ciency in SDS-CGE, especially when the proteins exist as distributions of derivatives, tends to be poor relative to CZE. Furthermore, there is a lack of data providing peak height or peak area reproducibility for £uorescently derivatized proteins
separated by SDS-CGE. Without adequate reproducibility, quantitative protein separations are not possible. 3.6. Solid phase labeling
Solid phase labeling is an interesting strategy to circumvent probe concentration requirements. Dovichi's group have used an Immobilon CD membrane to immobilize insulin chain B ( Ins B ) in an in-house constructed solid phase reactor [ 30 ]. Ins B can then be labeled with vast excess amounts of £uorescent probe, in this case FQ, without interference from £uorescent products due to hydrolysis and secondary reactions. These unwanted products are £ushed away before extraction of Ins B with low pH buffer and off-line analysis by micellar electrokinetic chromatography. This system was able to derivatize 1038 M Ins B
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by using a preconcentration step in the solid phase reactor. While this technique works for Ins B with only two possible derivatization sites, any protein labeled by this technique will yield multiple £uorescent derivatives. Krull's group has developed a number of methodologies for the derivatization of proteins for analysis by HPLC. These methodologies rely on solid phase labeling where the £uorescent derivatizing reagent, 6-aminoquinoline carbamate ( 6-AQC ), is immobilized onto a styrene^divinylbenzene support. The immobilized £uorophore possesses an activated linkage that will allow the nucleophilic addition of the 6AQC to a protein as it £ows past [ 31 ]. Using a speci¢c type of support with well-controlled physical properties, they have performed size selective derivatizations of proteins with selective labeling of only those primary amine( s ) that can access the interior of the support pores [ 32 ]. These methodologies may be of practical use for CE separations also, as the authors suggest, although it may prove that the signi¢cantly improved separation ef¢ciency of CE may partially resolve a distribution of products that HPLC can not.
4. Summary Although £uorescent derivatization of proteins for capillary electrophoretic separations is problematic, a number of useful methodologies have been described in this review. Each has its advantages and disadvantages, which suggests that there is room for improvement. A number of research groups are examining new ways of performing £uorescent derivatization for practical detection of proteins by CE. Krull's group is currently investigating the use of excess amounts of £uorescent probe in derivatization reactions in order to force complete derivatization [ 33 ]. The stoichiometric excess of probe must be relative to the number of primary amines in the analyte protein. In addition, it appears that the protein must be denatured to expose all groups to the probe. As one would expect, this technique is protein dependent in that some analytes require different degrees of denaturation to expose all N-termini and lysine residues to possible derivatization. Excellent results have been achieved for insulin where MALDI^TOF experiments have con¢rmed the derivatization of both N-termini and the lone lysine residue. The CZE separation of the derivatization reaction mixture demonstrates a unique insulin derivative and probe hydrolysis products. Furthermore, the sep-
aration ef¢ciency of the derivative is improved signi¢cantly relative to the native form [ 33 ]. The complete derivatization of other proteins is currently being examined. Although low concentration derivatization has not so far been investigated, the succinimidyl ester amine-reactive moiety used in this study has the ability to derivatize amino acids at nanomolar concentrations [ 10 ], thus the capability to derivative low protein concentrations could follow. Our group is currently investigating the use of the enzyme transglutaminase for the labeling of glutamine residues in proteins with a £uorescent, uncharged derivative of cadaverine ( for example: tetramethylrhodamine^cadaverine ). In this method ( Fig. 8 ), an uncharged residue is derivatized with an uncharged £uorophore which will provide a distribution of derivatives with the same charge and thus similar electrophoretic mobilities. Furthermore, the need for the enzyme should narrow the distribution of products generated in the derivatization reaction since presumably the protein to be derivatized must access the active site of the enzyme. One can anticipate steric hindrances limiting the number of glutamine residues that can be derivatized. If the probe concentration requirements are high with this method, the derivative could be used as an immune reagent for the competitive immunoassay of the native protein, as described by Kennedy's group.
5. Abbreviations ANS 6-AQC BSA CE CZE FITC FQ FTC hGH HPLC IgG Ins B Vex LIF LOD NBD-Cl NBD-F NDA OPA SDS SDS-CGE TNS
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1-anilinonaphthalene-8-sulfonate 6-aminoquinoline carbamate bovine serum albumin capillary electrophoresis capillary zone electrophoresis £uorescein isothiocyanate 5-furoylquinoline-3-carboxaldehyde £uorescein thiocarbamyl human growth hormone high performance liquid chromatography immunoglobulin G insulin, chain B wavelength of maximum excitation laser-induced £uorescence limit of detection 4-nitrobenz-2-oxa-1,3-diazole 4-£uoro-7-nitrobenzofurazan naphthalene-2,3-dicarboxaldehyde o-phthalaldehyde sodium dodecyl sulfate sodium dodecyl sulfate^capillary gel electrophoresis 2-p-toluidinonaphthalene-6-sulfonate
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References [ 1 ] Various authors, in J.P. Landers ( Editor ), The CRC Handbook of Capillary Electrophoresis, 2nd Edition, CRC Press, Boca Raton, FL, 1997. [ 2 ] A. Kunkel, M. Degenhardt, B. Schirm, H. Waëtzig, J. Chromatogr. A 768 ( 1997 ) 17. [ 3 ] D.N. Heiger, High Performance Capillary Electrophoresis ^ An Introduction, Hewlett-Packard Co. Publication Number 12-5091-6199E, p. 100. [ 4 ] S.E. Moring, R.T. Reel, R.E.J. van Soest, Anal. Chem. 65 ( 1993 ) 3454. [ 5 ] HP3D CE High Sensitivity Cell, Hewlett-Packard Co. Publication Number 12-5965-5984E. [ 6 ] D.F. Swaile, M.J. Sepaniak, J. Liq. Chromatogr. 14 ( 1991 ) 869. [ 7 ] T.T. Lee, E.S. Yeung, J. Chromatogr. 595 ( 1992 ) 319. [ 8 ] D.M. Paquette, R. Sing, P.R. Banks and K.C. Waldron, J. Chromatogr. B, ( 1998 ) in press. [ 9 ] Y.-F. Cheng, N.J. Dovichi, Science 242 ( 1988 ) 562. [ 10 ] S.K. Lau, F. Zaccardo, M. Little, P. Banks, J. Chromatogr. A 809 ( 1998 ) 203. [ 11 ] N. Klonis, W.H. Sawyer, J. Fluoresc. 6 ( 1996 ) 147. [ 12 ] P.R. Banks, D.M. Paquette, J. Chromatogr. A 693 ( 1995 ) 145. [ 13 ] I.S. Krull, R. Strong, Z. Sosic, B.-Y. Cho, S.C. Beale, C.-C. Wang, S. Cohen, J. Chromatogr. B 699 ( 1997 ) 173. [ 14 ] O. Orwar, M. Sandberg, I. Jacobson, M. Sundahl, S. Folestad, Anal. Chem. 67 ( 1995 ) 4261. [ 15 ] T. Ueda, R. Mitchell, F. Kitamura, T. Metcalf, T. Kuwana, A. Nakamoto, J. Chromatogr. 593 ( 1992 ) 265. [ 16 ] B. Nickerson, J.W. Jorgenson, J. Chromatogr. 480 ( 1989 ) 157. [ 17 ] L. Zhang, E.S. Yeung, J. Chromatogr. A 734 ( 1996 ) 331. [ 18 ] M.E. Szulc, I.S. Krull, J. Chromatogr. A 659 ( 1994 ) 231. [ 19 ] N.M. Schultz, R.T. Kennedy, Anal. Chem. 65 ( 1993 ) 3161. [ 20 ] N.M. Schultz, L. Huang, R.T. Kennedy, Anal. Chem. 67 ( 1995 ) 924. [ 21 ] N. Chiem, D.J. Harrison, Anal. Chem. 69 ( 1997 ) 373. [ 22 ] O.W. Reif, R. Lausch, T. Scheper, R. Freitag, Anal. Chem. 66 ( 1994 ) 4027.
[ 23 ] K. Shimura, B.L. Karger, Anal. Chem. 66 ( 1994 ) 9. [ 24 ] D. Schmalzing, W. Nashabeh, Electrophoresis 18 ( 1997 ) 2184. [ 25 ] D.M. Pinto, E.A. Arriaga, D. Craig, J. Angelova, N. Sharma, H. Ahmadzadeh, C.A. Boulet, N.J. Dovichi, Anal. Chem. 69 ( 1997 ) 3015. [ 26 ] E.L. Gump, C.A. Monnig, J. Chromatogr. A 715 ( 1995 ) 167. [ 27 ] E.T. Wise, N. Singh, B.L. Hogan, J. Chromatogr. A 746 ( 1996 ) 109. [ 28 ] C.-C. Wang, S.C. Beale, J. Chromatogr. A 756 ( 1996 ) 245. [ 29 ] M.D. Harvey, D. Bandilla and P.R. Banks, Electrophoresis, ( 1998 ) in press. [ 30 ] D.M. Pinto, E.A. Arriaga, S. Sia, Z. Li, N.J. Dovichi, Electrophoresis 16 ( 1995 ) 534. [ 31 ] G. Li, J. Yu, I.S. Krull, S. Cohen, J. Liq. Chromatogr. 18 ( 1995 ) 3889. [ 32 ] M.E. Szulc, P. Swett, I.S. Krull, Biomed. Chromatogr. 11 ( 1997 ) 207. [ 33 ] B.-Y. Cho, H. Liu and I.S. Krull, unpublished results. Peter R. Banks was born a British citizen in Bombay (Mumbai ), India in 1959. In 1961, he emigrated to Canada with his parents and became a Canadian citizen in 1965. He received a BSc in Chemistry from the University of British Columbia in 1986 and a PhD in Analytical Chemistry from the same institution in 1992 under the guidance of Michael W. Blades. His postdoctoral studies in the laboratories of Norman J. Dovichi at the University of Alberta were funded by an NSERC Postdoctoral Fellowship awarded for 1992^1994. Dr. Banks is currently an Assistant Professor in the Department of Chemistry and Biochemistry at Concordia University where he has been since 1994. His main research interests are directed towards developing methodology for the low concentration analysis of proteins, especially in clinical samples; the analysis of neurotransmitters from microdialysate samples using £uorescent derivatization; and the use of non-aqueous capillary electrophoresis for the analysis of non-polar analyte.
TrAC / Internet column In order to inform analytical chemists about the Internet and the role it could play in their lives, Dr. Michael Guilhaus was invited to become a Contributing Editor of TrAC. The Internet Column has now become a feature of the journal. The column can also be found on the World Wide Web. Anyone interested in contributing to this column is invited to contact Michael Guilhaus at: Mike^[email protected] The Internet Column articles of TrAC can also be found on the Web. If you have a browser, to access the TrAC column on the Web simply point to: http: / www.elsevier.nl / locate / trac
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