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The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries
Journal of Chromatography A, 1216 (2009) 3613–3620
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries Anisa Elhamili a , Magnus Wetterhall a , Angel Puerta a , Douglas Westerlund b , Jonas Bergquist a,∗ a b
Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University, Biomedical Center, P.O. Box 599, SE-751 24 Uppsala, Sweden Analytical Pharmaceutical Chemistry, Uppsala University, Biomedical Center, P.O. Box 574, SE-751 23 Uppsala, Sweden
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Article history: Available online 25 December 2008 Keywords: Capillary electrophoresis Alkali salt Intact protein analysis Coated capillary Stacking effect
a b s t r a c t The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1 × 106 apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Capillary electrophoresis (CE) has rapidly become a valuable tool in the analysis of peptides/proteins due to its short separation times, small injection volumes, high peak efficiencies and ease of automation [1]. However, the efficiency and precision of protein separations in CE may be severely compromised due to adsorption of the analytes to the capillary wall [1–5]. Thus, it is difficult to separate proteins without altering the chemistry at the silica wall to minimize these interactions. In uncoated fused silica capillaries, efficient protein separations have been performed by using different strategies. In some instances, the adjustment of the pH of the running buffer to extreme pH values [2,6–11] has been successful. However, a number of problems may arise such as protein aggregation and/or precipitation [2]. Other alternatives are the use of high-ionic strength buffers [2,12,13], or buffer additives such as amines, low conductive zwitterions [14] or alkali metal salts [15]. The more general approach to prevent protein adsorption is to use surface modified capillaries. Surface modifications can be performed either by covalent coupling to the silanol groups on the capillary surface [16–22] or by non-covalent modification using an adsorbed cationic surfactant or polymer [18,21,23–25]. Moreover, capillary coatings are the most preferred strategies when mass spectrometry (MS) is used as detection technique, since the other strategies are often not compatible with the MS detection [26,27]. A recent study performed by Elhamili et al. [28] showed that
∗ Corresponding author. Tel.: +46 18 471 3675; fax: +46 18 471 3692. E-mail address: [email protected] (J. Bergquist). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.12.037
the use of a monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza1-azoniabicyclo(2,2,2)octane iodide (M7C4I), for capillary coating provided high efficient separations (up to 1.8 × 106 plates/m) in the analysis of peptides, proteins and complex samples by CE–MS. Furthermore, that was the first time such a coating provided good peak shapes of extremely large proteins (669 kDa) analyzed by CE. The highest peak efficiency values were obtained when alkali salts were added to the protein sample. Similar salt effects have been reported by Shihabi et al. [29–34] showing a high degree of stacking when both acetonitrile and salts were added to the sample for the analysis of small molecules such as peptides in biological fluids. They reported that adding acetonitrile or a mix of acetonitrile and salt to the sample generates a larger stacking effect than adding salt alone since acetonitrile stops the enzymatic activity, remove proteins and improve the sensitivity and resolution. However, the conductivity in the sample plug was lower than for the background electrolyte (BGE) and the stacking effects can be explained due to the differences in conductivities. In this study, only salts were added to the sample and the conductivity in the sample plug was most often higher than in the BGE. Usually, the presence of high ionic strength buffers or samples deteriorates the CE separation and causes band broadening and poor separation efficiencies. This is due to “inverse stacking” and to development of Joule heating [35]. Therefore, the surprisingly positive influence of adding alkali salts to the sample on the peak efficiency of the CE analysis of proteins of different sizes (molecular weight (MW): 14–669 kDa) was further investigated in this work. The experiments were primarily carried out with M7C4I coated capillaries and then further evaluated with other coatings yielding positively charged or neutral surfaces.
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2. Experimental 2.1. Chemicals and reagents All chemicals were of analytical-reagent grade, and all solutions were prepared in ultra pure Milli-Q water (Milli-Q water system, Millipore, Bedford, MA, USA). Sodium hydroxide (NaOH), sodium chloride (NaCl), sodium iodide (NaI), hydrochloric acid (HCl), toluene and potassium chloride (KCl) were obtained from (Merck KGaA, Darmstadt, Germany). Acetic acid (HAc), ammonium acetate (NH4 Ac), ammonia solution 25% (NH3 ), formic acid (HCOOH) (Riedel-de Haen, Germany), and ammonium formate (NH4 COOH) from BDH (Poole, England), phosphoric acid (H3 PO4 ) (Merck) and ammonium di-hydrogen phosphate (NH4 H2 PO4 ) from Sigma–Aldrich–Chemie GmbH (Steinheim, Germany) were used for CE buffer preparation. The buffers were filtered by a Minisart N syringe filtration unit with filters of a pore size of 0.45 m (Sartorius AG, Göttingen, Germany). The M7C4I monomer, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo(2,2,2)octane iodide, for capillary coating was used as received from Righetti’s group [21,22]. Borate buffer (25 mM at pH 9.8) for capillary coating was obtained from Agilent Technologies (Waldbronn, Germany). N-Trimethoxysilyl-propylN,N,N-trimethyl-ammonium chloride (TAC) [19] monomer, was purchased from ABCR (Karlsruhe, Germany). PolyE-323 polymer for capillary coating was previously developed in our laboratory [23,25]. Polyvinylalcohol (PVA) coated capillaries (50 m inner diameter and 365 m outer diameter) were obtained from Agilent Technologies and used as recommended by the manufacturer. Untreated fused silica capillaries with 50 m inner diameter and 365 m outer diameters were obtained from Polymicro Technologies (Phoenix, AZ, USA). The proteins; lysozyme (MW: 14.3 kDa, pI: 9.6), ␣-chymotrypsinogen A (MW: 25.7 kDa, pI: 8.5), bovine serum albumin (BSA) (MW: 66 kDa, pI: 5.8), transferrin (MW: 80 kDa, pI: 6.8), ␥-globulin (MW: 165 kDa, pI: 8.5) and thyroglobulin (MW: 669 kDa, pI: 6.9) were purchased as lyophilized powder from Sigma (St. Louis, Mo, USA) and used as received without further purification. Stock solutions of proteins were prepared in Milli-Q water at the concentration of 1–5 mg/mL and stored at −20 ◦ C. Samples were diluted in either Milli-Q water or 10 mM ammonium formate pH 3.0 with salts at different concentrations. 2.2. Instrumentation The CE experiments were performed using an automated Agilent3D capillary electrophoresis system (Palo Alto, CA, USA) interfaced with a HP Pentium II PC. The separations were carried out at 25 ◦ C at negative field strengths of 250–450 V/cm with 10 s ramping (positive field strengths when using PVA coated capillaries) and the samples were hydrodynamically injected (50 mbar for 30 s corresponding to approximately 50 nL). All injections were carried out in triplicates or more and the capillaries were equilibrated for 2 min before each run with running buffer. Prior to the coating process, a 0.25 cm optical window was prepared on the fused-silica capillaries by using an optical fiber splicer (Model PFS 200 series; Power Technology, Little Rock, AR, USA). For CE–UV detection, the system was equipped with a photodiode-array detector and measurements were made at 200 nm for protein analysis. The total and effective lengths of the capillaries were 50 cm and 41.5 cm, respectively. The efficiencies were calculated by the statistical moment method provided by the standard Agilent software. 2.3. Procedure 2.3.1. M7C4I coating The coating procedure has been described in detail previously by Elhamili et al. [28]. The capillaries were rinsed (at 935 mbar) with
0.1 M NaOH for 40 min, followed by Milli-Q water (at 935 mbar) for 30 min, to activate the surface and to generate as many free silanols as possible before derivatization. This was followed by flushing the capillaries for 2 min (at 935 mbar) with the modifier solution 4 mM M7C4I in 25 mM borate buffer pH 9.8. Finally, the capillaries were rinsed (at 935 mbar) with the running buffer (10 mM ammonium formate pH 3.0) for 2 min to flush out excess of modifier solution, followed by the application of a negative voltage (−20 kV) for equilibration 3 min. The coating process was tested by injection of thiourea (Merck) dissolved in Milli-Q water (1 mg/mL, detection at 214 nm) at 50 mbar for 5 s. 2.3.2. TAC coating The TAC coating was performed according to Johannesson et al. [19]. The capillaries were first pre-treated with 6 M hydrochloric acid for 2 h, flushed with toluene for 30 min and dried for another 30 min with nitrogen. The monomer solution consisting of 5% TAC in toluene was flushed through the capillaries for 4 h, followed by a rinsing step with toluene for 30 min and drying with nitrogen for 30 min. The applied pressure was 2.0 bar. The coating process was tested by injection of thiourea. 2.3.3. PolyE-323 coating The PolyE-323 coating procedure has previously been described by our group [23,25]. In the pre-treatment step, the surface of a fused silica capillary was activated by rinsing with 1 M NaOH at 1 bar for 30 min, followed by flushing with Milli-Q water for 5 min. After the pre-treatment steps, the capillaries were flushed for 10 min with PolyE-323 (7.5%, w/w) adjusted to pH 7 with acetic acid. Excess polymer solution was then washed out with 50 mM ammonium acetate buffer, pH 5, for 5 min. The coating process was tested by injection of thiourea. 3. Results and discussions 3.1. Effect of salt concentration on the peak efficiency using M7C4I coatings As shown in a previous study [28], we were able for the first time to run and get high peak efficiencies of extremely large proteins (669 kDa) by CE with M7C4I derivatized silica capillaries. Unexpectedly, the highest efficiencies were obtained with alkali salts added to the sample. Shihabi et al. have previously shown that adding sodium chloride and acetonitrile in the sample will lead to sample stacking effects and increased resolution in the CE separation [29–34]. However, a major difference in those studies compared to Elhamili et al. was the fact that the ionic strength (and conductivity) in the BGE was higher than in the sample and the stacking was dependent on the presence of both salts and acetonitrile in the sample. Thus, in the present work, the effect of adding alkali salts at different concentrations to the sample was investigated regarding the peak efficiency of proteins of different sizes. The ionic strength in the BGE was kept constant at 10 mM, while the ionic strength of the salt in the sample was varied between 0–100 mM and therefore, most often higher than in the BGE. Moreover, no organic modifiers were added to the sample. Despite the higher ionic strength in the sample compared to the BGE, a dramatic peak stacking effect was observed for the CE analysis of proteins with molecular weights ranging from 14 kDa up to 669 kDa. The efficiencies discussed in the paper are the apparent efficiencies, which is justified in comparisons using the same type of coated capillary, since the electroosmosis was not influenced by the variation of salt concentrations in the sample. However, for comparisons of efficiencies between different types of capillaries when the electroosmosis differs true efficiencies should be used [36]. Fig. 1A–F shows a set of peak patterns for the proteins analyzed in the presence of different
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Fig. 1. CE–UV analysis of proteins with MW range of 14–669 kDa on M7C4I coated capillary as a function of the concentration of NaCl (0–100 mM) (increase by 10 mM) in the protein sample. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; 1.5 M protein solution (0.25 M for thyroglobulin) was injected at 50 mbar for 30 s (injected volume 50 nL); −25 kV applied voltage; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more. (A) Lysozyme, (B) ␣-chymotrypsinogen, (C) BSA, (D) transferrin, (E) ␥-globulin, and (F) thyroglobulin.
concentration of NaCl (0–100 mM). The obtained results showed that the increased concentration of salt in the protein samples provided a sharpening of the protein peaks. The addition of NaCl promoted a high degree of peak stacking with very high efficiencies (up to 2.1 × 106 plates/m). High ionic strengths in the background electrolyte have been reported to prevent adsorption of proteins to the capillary surface [14,15], but not providing such stacking effects as observed here. The obtained results might support the hypothe-
sis that the increased ionic strength would compact the layer of the M7C4I coating on the silica wall and thus avoiding or minimizing the electrostatic interactions between the proteins and the silanols on the capillary surface. If this hypothesis would be the major cause for the peak sharpening, then flushing the capillaries with NaCl solution prior to analysis would yield the same effect as adding the salt to the sample plug. However, this approach, which involved flushing the capillaries with (1:1, v/v) 0.5 M NaCl in the BGE instead
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Fig. 3. Peak efficiencies for different molar concentrations of ␥-globulin as a function of salt concentration in the sample solution using M7C4I coated capillaries. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window. Concentration of ␥-globulin: 0.25, 0.5, 0.75, and 1.5 M was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3; approximately 50 nL of the sample was injected. The data is the average of triplicate injections with the CV for efficiency ranging from 0.01 to 1.8% for each molar concentration of ␥-globulin.
Fig. 2. (A) CE peak efficiencies of ␥-globulin, transferrin, BSA and lysozyme and (B) efficiency of ␣-chymotrypsinogen as function of NaCl concentrations added to the sample on M7C4I coated capillaries. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; 1.5 M protein solution was injected at 50 mbar for 30 s (injected volume 50 nL); −25 kV applied voltage; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more The CV for the peak efficiencies was ranging from 0.01 to 2.3% for each protein.
of dissolving the protein samples in NaCl, did not improve the efficiency as when only adding salt to the sample. Moreover, adding salt to the running buffer to create a stacking effect will eventually cause Joule heating and peak broadening. Thus, the amount of salt that can be added to the buffer is rather limited compared to what can be added to the small sample plug. One interesting observation is that the concentration of NaCl needed to yield peak sharpening for each protein seems to be dependent on the molecular weight of each protein. The optimum molar NaCl concentrations in the protein sample needed to obtain the best peak efficiency for ␥-globulin (165 kDa), transferrin (80 kDa), BSA (66 kDa), lysozyme (14.3 kDa) were 70 mM, 50 mM, 20 mM and 10 mM, respectively (Fig. 1). Fig. 2A shows the peak efficiency of each protein plotted as a function of the NaCl concentration (CV for efficiency ranging from 0.01 to 2.3% based on triplicate injections for each protein), where the highest efficiencies obtained at the optimum salt concentration for ␥-globulin, transferrin, BSA and lysozyme were 3.9 × 105 plates/m, 3.8 × 105 plates/m, 8.5 × 104 plates/m and 2.9 × 105 plates/m, respectively. One possible explanation to the observations could be an pseudo isotachophoretic phenomenon caused by the added alkali salts, which is consistent with previous observations by Shihabi et al. [29,34]. The obtained results could also be due to increased protein charging by the added alkali ions.
Large MW proteins have the possibility to bind a higher amount of alkali ions than small MW proteins. However, if a sufficient molar concentration of salt is added to the protein sample, the protein will eventually reach its maximum charge-to-size-ratio and no more alkali ions can be bound to the protein. Thus, the chargeto-size ratio (and migration times) would be very similar for all proteins regardless of their molecular weight. One studied protein, ␣-chymotrypsinogen, does not follow the observed molecular weight trend. This protein requires a fairly high concentration of NaCl (60 mM) (as it can be seen in Figs. 1B and 2B) to get the highest efficiency (2.1 × 106 plates/m) although it has a MW of approximately 26 kDa (CV for efficiency ranging from 0.01 to 1.3% based on triplicate injections). Moreover, there was shift in the migration time of this protein when NaCl concentration in the sample was higher than 40 mM as can be seen in Fig. 1B. The reason for this deviation is not known, but might be due to a conformation change at a critical salt concentration. To further investigate the possible effect of protein charging, the efficiency for the protein ␥-globulin, injected at different molar concentrations, was plotted against the molar concentration of NaCl added to the sample (Fig. 3). It can clearly be seen that an increasing protein concentration requires an increasing concentration of salt added to the sample to obtain the maximum peak efficiency (CV for efficiency ranging from 0.01 to 1.8% based on triplicate injections for each molar concentration of ␥-globulin). These results are supporting the hypothesis of a protein charging effect. To investigate if there was any difference in dissolving the protein in MQ-water and salt, where the protolytic properties of the protein determines the pH, or in the BGE and salt, ␥-globulin was tested with M7C4I coated capillaries. The peak efficiencies and peak heights increased (3.5 × 102 plates/m to 3.9 × 105 plates/m and 20 mAU to 369 mAU respectively for MQ–NaCl and 2.9 × 102 plates/m to 3.1 × 105 plates/m and 18 mAU to 307 mAU respectively for BGE–NaCl) with increasing NaCl concentrations (data not shown). The maximum values for the peak efficiencies and peak heights were reached at 70 mM NaCl, regardless if the protein was dissolved in MQ–NaCl or BGE–NaCl. Thus, the observed peak sharpening effect is not dependent on the pH or buffering capacity in the sample plug and occurs both in buffered and unbuffered sample plugs. Similar results were obtained for the other proteins (data not shown) which implies that the observed phenomenon is not protein specific.
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for NaI compared to NaCl but at NaI concentrations above 40 mM there was a dramatic increase in peak efficiency up to the highest value 1.3 × 106 plates/m at 70 mM NaI and then a radical decrease (CV for efficiency ranging from 0.01 to 2.0% for NaCl and 0.02 to 2.4 for NaI based on triplicate injections). When NaCl was added, there was gradual increase in the efficiencies and the maximum value was also at 70 mM. One major drawback in using NaI is the presence of an interfering iodide peak in the electropherograms, when using UV detection (data not shown). There is a general improvement of the peak efficiency, regardless of which alkali salt added to the sample. However, the underlying reason for this needs to be further evaluated. 3.3. Salt effect on the separation of a mixture of proteins using M7C4I coated capillaries
Fig. 4. (A) Peak efficiencies of transferrin analyzed at different concentrations of NaCl and KCl added to the sample and (B) peak efficiencies of ␥-globulin at different concentration of NaCl and NaI added to the sample analyzed on M7C4I coated capillaries. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3; approximately 50 nL of the sample was injected. The data is the average of triplicate injections with the CV for efficiency ranging from 0.02 to 2.4% for transferrin, and 0.01 to 2.4% for ␥-globulin.
3.2. Effect of different alkali salts on the peak efficiency using M7C4I coatings Other alkali salts, potassium chloride (KCl) and sodium iodide (NaI), were added to the protein samples at identical conditions as for NaCl to evaluate if similar results could be obtained as for NaCl. The effect of different concentrations of the added salts (0–100 mM) was studied for the proteins transferrin and ␥-globulin. The results indicate that KCl has an improving effect on the peak efficiency of transferrin, however, not as strong as NaCl (Fig. 4A) (CV for efficiency ranging from 0.02 to 1.4% for NaCl and 0.03 to 2.4 for KCl based on triplicate injections). Furthermore, when KCl was added to 50–70 mM concentration to the sample, there was a sudden drop in the peak efficiency for transferrin. The obtained results indicate that the addition of alkali salts to the sample has a positive effect on the peak efficiency and that the effect of Na+ seems to be more pronounced than K+ . The effect of using another halogen than chloride was investigated by adding different concentrations of NaI to samples of ␥-globulin, see Fig. 4B. The obtained results showed that the peak efficiency can also be improved by NaI salt addition. However, the shape of the peak efficiency curves differs. At low salt concentration (up to 40 mM) somewhat lower efficiencies were obtained
Mixtures of four proteins (lysozyme, BSA, transferrin and ␥globulin) in equal molar concentrations (1.5 M) were dissolved in different concentrations of NaCl and analyzed to study the effect of salt additions on the resolution of the separation. The resolution in the separation increased when low molar concentrations of NaCl were added to the sample compared to separation of proteins dissolved in pure MQ-water (Fig. 5). However, as the added salt concentration exceeds 15–20 mM NaCl, the proteins mixture start to co-migrate into an even sharper peak. At higher concentration than 20 mM, no separation of the proteins could be observed. A possible explanation to the co-migration at high salt concentrations could be that all proteins, regardless of their mass, will reach the same, or very similar charge-to-size ratio due to the excess of salt and thus the same electrophoretic migration. To evaluate this, the migration times for the individually injected proteins was normalized against the EOF (calculated by the water dip in the electropherograms) and plotted in Fig. 6. The normalized values all reach the same value above 40 mM NaCl, except for lysozyme. This supports the hypothesis of protein charging to reach the same charge-to-size ratio, regardless of MW of the protein. 3.4. Effect of salt concentration on the peak efficiency using PolyE-323 surfaces To investigate if the effect of the improvement of the efficiency in the separation when adding salt to the sample was surface coating dependent, other coatings were investigated. The PolyE-323 [23,25] polymer interacts electrostatically with the ionized silanol groups and as it is a polycationic polymer it yields a positive surface with reversed EOF, similar to the M7C4I coated silica capillaries. This coating has previously been developed by our group and has shown to be suitable for bioanalytical applications [23,25]. The analysis of transferrin at different NaCl concentrations (0–100 mM) is shown in Fig. 7. The peak intensity and efficiencies were much lower compared to the covalent M7C4I coating and no peak stacking effects could be observed with increasing salt concentration. One possible explanation could be that presence of salt reduces the electrostatic interactions between PolyE-323 and the silica wall. However, that would have reduced the EOF at higher salt concentrations, which was not observed. 3.5. Effect of salt concentration on the peak efficiency using TAC surfaces Another covalent monomer coating was tested to evaluate if the M7C4I results could be obtained with a different positively charged monomer coating. For this purpose, analyses were carried out using the TAC monomer for silica surface modification. The TAC coating attaches covalently to the silanol groups, producing a stable, positively charged surface and reversed EOF [19]. The
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Fig. 6. Normalized migration times for the proteins lysozyme, ␣-chymotrypsinogen transferrin and ␥-globulin as a function of salt concentration added to the protein sample. The migration times were normalized against the water dip in the electropherrograms. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more.
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Fig. 8. CE–UV analysis of transferrin on TAC coated capillary as a function of the NaCl concentration (0–100 mM) in the protein sample. The transferrin concentration was 1.5 M. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more.
bic properties than M7C4I, which could also partly explain the results. 3.6. Effect of salt concentration on the peak efficiency using neutral PVA surfaces
Fig. 7. CE–UV analysis of transferrin by adding different concentrations of NaCl (0–100 mM) to the sample on PolyE-323 coated capillaries. The protein concentration was 1.5 M. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more.
influence of adding different NaCl concentrations in a transferrin sample was studied. The highest peak efficiency for transferrin was obtained at a much lower salt concentration (10 mM) compared to what was needed to get the maximum efficiency with the M7C4I coating (50 mM) as shown in Fig. 8. A possible explanation for the lower salt concentration needed for optimal efficiencies on the TAC coating could be related to a higher charge density obtained with this coating due to the smaller molecular size of the coating reagent. This assumption is supported by the observed higher EOF obtained for the TAC coating compared to the M7C4I. Furthermore, the TAC coating reagent has less hydropho-
The M7C4I, PolyE-323 and TAC coatings all generate a positively charged surface with high electroosmotic flows. The positive surface for these coatings will have electrostatic repulsion effects on the proteins, especially when a low pH is used in the BGE and sample plug and when an alkali salt is added to the sample. PVA coated capillaries have a hydrophilic and neutral surface, thus generating no EOF in CE analysis. These capillaries are commercially available and readily used for protein and peptide separation with CE [37,38]. Therefore, PVA coated capillaries were tested to investigate if the observed peak stacking effect could be obtained with a neutral surface. Transferrin dissolved in Milli-Q water and different NaCl concentrations (0–100 mM) or dissolved in BGE (10 mM ammonium formate pH 3.0) and different NaCl concentrations (0–100 mM) were studied. The peak efficiencies and peak heights increased (9.2 × 103 plates/m to 1.7 × 106 plates/m and 21 mAU to 440 mAU respectively for Milli-Q–NaCl and 8.7 × 103 plates/m to 1.1 × 106 plates/m and 17 mAU to 413 mAU respectively for BGE–NaCl) with increasing NaCl concentrations (data not shown). A difference to the positively charged surfaces was that the highest peak efficiencies were reached at the highest NaCl-concentration tested, i.e., no maximum value as observed for the other coatings was obtained. Interestingly, no difference between dissolving transferrin in MQ–NaCl or BGE–NaCl could be observed, which is consistent with the results for the M7C4I coating and showing that the effect is obtained also with a proper pH-control of the introduced sample. 4. Conclusions The effect of adding different concentrations of alkali salts to protein samples with a large range of molecular weight
Fig. 5. CE–UV separations of a mixture of the proteins lysozyme, BSA, transferrin and ␥-globulin on M7C4I coated capillary as a function of the NaCl concentration added to the protein sample. The concentration of each protein in the sample was 1.5 M. Other conditions; capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3; approximately (A) MQ water, (B) 3 mM NaCl, (C) 5 mM NaCl, (D) 10 mM NaCl, (E) 15 mM NaCl, (F) 20 mM NaCl, (G) 30 mM NaCl and (H) 70 mM NaCl.
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(14–669 kDa) analyzed by CE with various surface modified capillaries was conducted in this study. Regardless of molecular weight, a dramatic improvement in peak efficiency and peak height was observed when adding the alkali salts to the protein sample except for the PolyE-323 polymer coated capillaries. This coating is not covalently bound to the silica surface. It is electrostatically attracted to the silanols, which is a major difference compared to the other surfaces investigated. The optimal concentration of alkali salts needed in the sample plug is dependent on protein MW and molar concentration of the protein, indicating a protein–salt interaction phenomenon. In addition, the optimal salt concentration varies depending on the surface coating used implying that the results also are due to salt–surface interactions. These interactions will differ depending on the nature of the surface and thus explain the observed differences between the different coatings. Therefore, based on the obtained results with the M7C4I coating, in combination with PolyE-323, TAC and PVA, it is highly likely that the observed peak sharpening effect when adding salt to the sample is a mixed effect of both protein–salt and salt–surface interactions. Adding salts to a protein mixture will initially increase the peak efficiencies and resolution. However, an excess of salt will cause all proteins in the mixture to co-migrate in one sharp peak and thereby ruin the separation. A possible explanation for this is that the proteins will reach very similar charge-to-size ratios and therefore not separated in the CE analysis. In this study, we are reporting the interesting and somewhat surprising peak stacking effect for proteins when alkali salts were added to the sample. The underlying mechanism behind the observed effect is still unclear. Therefore, further studies regarding this phenomenon are planned and will be conducted. Acknowledgments This work was supported by a PhD grant from the Libyan government to Anisa Elhamili. A. Puerta acknowledges Spanish Ministry of Education and Science for a postdoctoral grant to perform research in Sweden. The Swedish Research Council is acknowledged for their support (grants, 629-2005-5379 and 629-2002-6821). The authors would like to thank Prof. Roberto Sebastiano and Prof. Pier Giorgio Righetti for supplying the M7C4I coating, and we would also like to acknowledge Prof. Jan Ståhlberg for additional fruitful discussions regarding the obtained results.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries Anisa Elhamili a , Magnus Wetterhall a , Angel Puerta a , Douglas Westerlund b , Jonas Bergquist a,∗ a b
Analytical Chemistry, Department of Physical and Analytical Chemistry, Uppsala University, Biomedical Center, P.O. Box 599, SE-751 24 Uppsala, Sweden Analytical Pharmaceutical Chemistry, Uppsala University, Biomedical Center, P.O. Box 574, SE-751 23 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Available online 25 December 2008 Keywords: Capillary electrophoresis Alkali salt Intact protein analysis Coated capillary Stacking effect
a b s t r a c t The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1 × 106 apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Capillary electrophoresis (CE) has rapidly become a valuable tool in the analysis of peptides/proteins due to its short separation times, small injection volumes, high peak efficiencies and ease of automation [1]. However, the efficiency and precision of protein separations in CE may be severely compromised due to adsorption of the analytes to the capillary wall [1–5]. Thus, it is difficult to separate proteins without altering the chemistry at the silica wall to minimize these interactions. In uncoated fused silica capillaries, efficient protein separations have been performed by using different strategies. In some instances, the adjustment of the pH of the running buffer to extreme pH values [2,6–11] has been successful. However, a number of problems may arise such as protein aggregation and/or precipitation [2]. Other alternatives are the use of high-ionic strength buffers [2,12,13], or buffer additives such as amines, low conductive zwitterions [14] or alkali metal salts [15]. The more general approach to prevent protein adsorption is to use surface modified capillaries. Surface modifications can be performed either by covalent coupling to the silanol groups on the capillary surface [16–22] or by non-covalent modification using an adsorbed cationic surfactant or polymer [18,21,23–25]. Moreover, capillary coatings are the most preferred strategies when mass spectrometry (MS) is used as detection technique, since the other strategies are often not compatible with the MS detection [26,27]. A recent study performed by Elhamili et al. [28] showed that
∗ Corresponding author. Tel.: +46 18 471 3675; fax: +46 18 471 3692. E-mail address: [email protected] (J. Bergquist). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.12.037
the use of a monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza1-azoniabicyclo(2,2,2)octane iodide (M7C4I), for capillary coating provided high efficient separations (up to 1.8 × 106 plates/m) in the analysis of peptides, proteins and complex samples by CE–MS. Furthermore, that was the first time such a coating provided good peak shapes of extremely large proteins (669 kDa) analyzed by CE. The highest peak efficiency values were obtained when alkali salts were added to the protein sample. Similar salt effects have been reported by Shihabi et al. [29–34] showing a high degree of stacking when both acetonitrile and salts were added to the sample for the analysis of small molecules such as peptides in biological fluids. They reported that adding acetonitrile or a mix of acetonitrile and salt to the sample generates a larger stacking effect than adding salt alone since acetonitrile stops the enzymatic activity, remove proteins and improve the sensitivity and resolution. However, the conductivity in the sample plug was lower than for the background electrolyte (BGE) and the stacking effects can be explained due to the differences in conductivities. In this study, only salts were added to the sample and the conductivity in the sample plug was most often higher than in the BGE. Usually, the presence of high ionic strength buffers or samples deteriorates the CE separation and causes band broadening and poor separation efficiencies. This is due to “inverse stacking” and to development of Joule heating [35]. Therefore, the surprisingly positive influence of adding alkali salts to the sample on the peak efficiency of the CE analysis of proteins of different sizes (molecular weight (MW): 14–669 kDa) was further investigated in this work. The experiments were primarily carried out with M7C4I coated capillaries and then further evaluated with other coatings yielding positively charged or neutral surfaces.
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2. Experimental 2.1. Chemicals and reagents All chemicals were of analytical-reagent grade, and all solutions were prepared in ultra pure Milli-Q water (Milli-Q water system, Millipore, Bedford, MA, USA). Sodium hydroxide (NaOH), sodium chloride (NaCl), sodium iodide (NaI), hydrochloric acid (HCl), toluene and potassium chloride (KCl) were obtained from (Merck KGaA, Darmstadt, Germany). Acetic acid (HAc), ammonium acetate (NH4 Ac), ammonia solution 25% (NH3 ), formic acid (HCOOH) (Riedel-de Haen, Germany), and ammonium formate (NH4 COOH) from BDH (Poole, England), phosphoric acid (H3 PO4 ) (Merck) and ammonium di-hydrogen phosphate (NH4 H2 PO4 ) from Sigma–Aldrich–Chemie GmbH (Steinheim, Germany) were used for CE buffer preparation. The buffers were filtered by a Minisart N syringe filtration unit with filters of a pore size of 0.45 m (Sartorius AG, Göttingen, Germany). The M7C4I monomer, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo(2,2,2)octane iodide, for capillary coating was used as received from Righetti’s group [21,22]. Borate buffer (25 mM at pH 9.8) for capillary coating was obtained from Agilent Technologies (Waldbronn, Germany). N-Trimethoxysilyl-propylN,N,N-trimethyl-ammonium chloride (TAC) [19] monomer, was purchased from ABCR (Karlsruhe, Germany). PolyE-323 polymer for capillary coating was previously developed in our laboratory [23,25]. Polyvinylalcohol (PVA) coated capillaries (50 m inner diameter and 365 m outer diameter) were obtained from Agilent Technologies and used as recommended by the manufacturer. Untreated fused silica capillaries with 50 m inner diameter and 365 m outer diameters were obtained from Polymicro Technologies (Phoenix, AZ, USA). The proteins; lysozyme (MW: 14.3 kDa, pI: 9.6), ␣-chymotrypsinogen A (MW: 25.7 kDa, pI: 8.5), bovine serum albumin (BSA) (MW: 66 kDa, pI: 5.8), transferrin (MW: 80 kDa, pI: 6.8), ␥-globulin (MW: 165 kDa, pI: 8.5) and thyroglobulin (MW: 669 kDa, pI: 6.9) were purchased as lyophilized powder from Sigma (St. Louis, Mo, USA) and used as received without further purification. Stock solutions of proteins were prepared in Milli-Q water at the concentration of 1–5 mg/mL and stored at −20 ◦ C. Samples were diluted in either Milli-Q water or 10 mM ammonium formate pH 3.0 with salts at different concentrations. 2.2. Instrumentation The CE experiments were performed using an automated Agilent3D capillary electrophoresis system (Palo Alto, CA, USA) interfaced with a HP Pentium II PC. The separations were carried out at 25 ◦ C at negative field strengths of 250–450 V/cm with 10 s ramping (positive field strengths when using PVA coated capillaries) and the samples were hydrodynamically injected (50 mbar for 30 s corresponding to approximately 50 nL). All injections were carried out in triplicates or more and the capillaries were equilibrated for 2 min before each run with running buffer. Prior to the coating process, a 0.25 cm optical window was prepared on the fused-silica capillaries by using an optical fiber splicer (Model PFS 200 series; Power Technology, Little Rock, AR, USA). For CE–UV detection, the system was equipped with a photodiode-array detector and measurements were made at 200 nm for protein analysis. The total and effective lengths of the capillaries were 50 cm and 41.5 cm, respectively. The efficiencies were calculated by the statistical moment method provided by the standard Agilent software. 2.3. Procedure 2.3.1. M7C4I coating The coating procedure has been described in detail previously by Elhamili et al. [28]. The capillaries were rinsed (at 935 mbar) with
0.1 M NaOH for 40 min, followed by Milli-Q water (at 935 mbar) for 30 min, to activate the surface and to generate as many free silanols as possible before derivatization. This was followed by flushing the capillaries for 2 min (at 935 mbar) with the modifier solution 4 mM M7C4I in 25 mM borate buffer pH 9.8. Finally, the capillaries were rinsed (at 935 mbar) with the running buffer (10 mM ammonium formate pH 3.0) for 2 min to flush out excess of modifier solution, followed by the application of a negative voltage (−20 kV) for equilibration 3 min. The coating process was tested by injection of thiourea (Merck) dissolved in Milli-Q water (1 mg/mL, detection at 214 nm) at 50 mbar for 5 s. 2.3.2. TAC coating The TAC coating was performed according to Johannesson et al. [19]. The capillaries were first pre-treated with 6 M hydrochloric acid for 2 h, flushed with toluene for 30 min and dried for another 30 min with nitrogen. The monomer solution consisting of 5% TAC in toluene was flushed through the capillaries for 4 h, followed by a rinsing step with toluene for 30 min and drying with nitrogen for 30 min. The applied pressure was 2.0 bar. The coating process was tested by injection of thiourea. 2.3.3. PolyE-323 coating The PolyE-323 coating procedure has previously been described by our group [23,25]. In the pre-treatment step, the surface of a fused silica capillary was activated by rinsing with 1 M NaOH at 1 bar for 30 min, followed by flushing with Milli-Q water for 5 min. After the pre-treatment steps, the capillaries were flushed for 10 min with PolyE-323 (7.5%, w/w) adjusted to pH 7 with acetic acid. Excess polymer solution was then washed out with 50 mM ammonium acetate buffer, pH 5, for 5 min. The coating process was tested by injection of thiourea. 3. Results and discussions 3.1. Effect of salt concentration on the peak efficiency using M7C4I coatings As shown in a previous study [28], we were able for the first time to run and get high peak efficiencies of extremely large proteins (669 kDa) by CE with M7C4I derivatized silica capillaries. Unexpectedly, the highest efficiencies were obtained with alkali salts added to the sample. Shihabi et al. have previously shown that adding sodium chloride and acetonitrile in the sample will lead to sample stacking effects and increased resolution in the CE separation [29–34]. However, a major difference in those studies compared to Elhamili et al. was the fact that the ionic strength (and conductivity) in the BGE was higher than in the sample and the stacking was dependent on the presence of both salts and acetonitrile in the sample. Thus, in the present work, the effect of adding alkali salts at different concentrations to the sample was investigated regarding the peak efficiency of proteins of different sizes. The ionic strength in the BGE was kept constant at 10 mM, while the ionic strength of the salt in the sample was varied between 0–100 mM and therefore, most often higher than in the BGE. Moreover, no organic modifiers were added to the sample. Despite the higher ionic strength in the sample compared to the BGE, a dramatic peak stacking effect was observed for the CE analysis of proteins with molecular weights ranging from 14 kDa up to 669 kDa. The efficiencies discussed in the paper are the apparent efficiencies, which is justified in comparisons using the same type of coated capillary, since the electroosmosis was not influenced by the variation of salt concentrations in the sample. However, for comparisons of efficiencies between different types of capillaries when the electroosmosis differs true efficiencies should be used [36]. Fig. 1A–F shows a set of peak patterns for the proteins analyzed in the presence of different
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Fig. 1. CE–UV analysis of proteins with MW range of 14–669 kDa on M7C4I coated capillary as a function of the concentration of NaCl (0–100 mM) (increase by 10 mM) in the protein sample. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; 1.5 M protein solution (0.25 M for thyroglobulin) was injected at 50 mbar for 30 s (injected volume 50 nL); −25 kV applied voltage; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more. (A) Lysozyme, (B) ␣-chymotrypsinogen, (C) BSA, (D) transferrin, (E) ␥-globulin, and (F) thyroglobulin.
concentration of NaCl (0–100 mM). The obtained results showed that the increased concentration of salt in the protein samples provided a sharpening of the protein peaks. The addition of NaCl promoted a high degree of peak stacking with very high efficiencies (up to 2.1 × 106 plates/m). High ionic strengths in the background electrolyte have been reported to prevent adsorption of proteins to the capillary surface [14,15], but not providing such stacking effects as observed here. The obtained results might support the hypothe-
sis that the increased ionic strength would compact the layer of the M7C4I coating on the silica wall and thus avoiding or minimizing the electrostatic interactions between the proteins and the silanols on the capillary surface. If this hypothesis would be the major cause for the peak sharpening, then flushing the capillaries with NaCl solution prior to analysis would yield the same effect as adding the salt to the sample plug. However, this approach, which involved flushing the capillaries with (1:1, v/v) 0.5 M NaCl in the BGE instead
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Fig. 3. Peak efficiencies for different molar concentrations of ␥-globulin as a function of salt concentration in the sample solution using M7C4I coated capillaries. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window. Concentration of ␥-globulin: 0.25, 0.5, 0.75, and 1.5 M was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3; approximately 50 nL of the sample was injected. The data is the average of triplicate injections with the CV for efficiency ranging from 0.01 to 1.8% for each molar concentration of ␥-globulin.
Fig. 2. (A) CE peak efficiencies of ␥-globulin, transferrin, BSA and lysozyme and (B) efficiency of ␣-chymotrypsinogen as function of NaCl concentrations added to the sample on M7C4I coated capillaries. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; 1.5 M protein solution was injected at 50 mbar for 30 s (injected volume 50 nL); −25 kV applied voltage; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more The CV for the peak efficiencies was ranging from 0.01 to 2.3% for each protein.
of dissolving the protein samples in NaCl, did not improve the efficiency as when only adding salt to the sample. Moreover, adding salt to the running buffer to create a stacking effect will eventually cause Joule heating and peak broadening. Thus, the amount of salt that can be added to the buffer is rather limited compared to what can be added to the small sample plug. One interesting observation is that the concentration of NaCl needed to yield peak sharpening for each protein seems to be dependent on the molecular weight of each protein. The optimum molar NaCl concentrations in the protein sample needed to obtain the best peak efficiency for ␥-globulin (165 kDa), transferrin (80 kDa), BSA (66 kDa), lysozyme (14.3 kDa) were 70 mM, 50 mM, 20 mM and 10 mM, respectively (Fig. 1). Fig. 2A shows the peak efficiency of each protein plotted as a function of the NaCl concentration (CV for efficiency ranging from 0.01 to 2.3% based on triplicate injections for each protein), where the highest efficiencies obtained at the optimum salt concentration for ␥-globulin, transferrin, BSA and lysozyme were 3.9 × 105 plates/m, 3.8 × 105 plates/m, 8.5 × 104 plates/m and 2.9 × 105 plates/m, respectively. One possible explanation to the observations could be an pseudo isotachophoretic phenomenon caused by the added alkali salts, which is consistent with previous observations by Shihabi et al. [29,34]. The obtained results could also be due to increased protein charging by the added alkali ions.
Large MW proteins have the possibility to bind a higher amount of alkali ions than small MW proteins. However, if a sufficient molar concentration of salt is added to the protein sample, the protein will eventually reach its maximum charge-to-size-ratio and no more alkali ions can be bound to the protein. Thus, the chargeto-size ratio (and migration times) would be very similar for all proteins regardless of their molecular weight. One studied protein, ␣-chymotrypsinogen, does not follow the observed molecular weight trend. This protein requires a fairly high concentration of NaCl (60 mM) (as it can be seen in Figs. 1B and 2B) to get the highest efficiency (2.1 × 106 plates/m) although it has a MW of approximately 26 kDa (CV for efficiency ranging from 0.01 to 1.3% based on triplicate injections). Moreover, there was shift in the migration time of this protein when NaCl concentration in the sample was higher than 40 mM as can be seen in Fig. 1B. The reason for this deviation is not known, but might be due to a conformation change at a critical salt concentration. To further investigate the possible effect of protein charging, the efficiency for the protein ␥-globulin, injected at different molar concentrations, was plotted against the molar concentration of NaCl added to the sample (Fig. 3). It can clearly be seen that an increasing protein concentration requires an increasing concentration of salt added to the sample to obtain the maximum peak efficiency (CV for efficiency ranging from 0.01 to 1.8% based on triplicate injections for each molar concentration of ␥-globulin). These results are supporting the hypothesis of a protein charging effect. To investigate if there was any difference in dissolving the protein in MQ-water and salt, where the protolytic properties of the protein determines the pH, or in the BGE and salt, ␥-globulin was tested with M7C4I coated capillaries. The peak efficiencies and peak heights increased (3.5 × 102 plates/m to 3.9 × 105 plates/m and 20 mAU to 369 mAU respectively for MQ–NaCl and 2.9 × 102 plates/m to 3.1 × 105 plates/m and 18 mAU to 307 mAU respectively for BGE–NaCl) with increasing NaCl concentrations (data not shown). The maximum values for the peak efficiencies and peak heights were reached at 70 mM NaCl, regardless if the protein was dissolved in MQ–NaCl or BGE–NaCl. Thus, the observed peak sharpening effect is not dependent on the pH or buffering capacity in the sample plug and occurs both in buffered and unbuffered sample plugs. Similar results were obtained for the other proteins (data not shown) which implies that the observed phenomenon is not protein specific.
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for NaI compared to NaCl but at NaI concentrations above 40 mM there was a dramatic increase in peak efficiency up to the highest value 1.3 × 106 plates/m at 70 mM NaI and then a radical decrease (CV for efficiency ranging from 0.01 to 2.0% for NaCl and 0.02 to 2.4 for NaI based on triplicate injections). When NaCl was added, there was gradual increase in the efficiencies and the maximum value was also at 70 mM. One major drawback in using NaI is the presence of an interfering iodide peak in the electropherograms, when using UV detection (data not shown). There is a general improvement of the peak efficiency, regardless of which alkali salt added to the sample. However, the underlying reason for this needs to be further evaluated. 3.3. Salt effect on the separation of a mixture of proteins using M7C4I coated capillaries
Fig. 4. (A) Peak efficiencies of transferrin analyzed at different concentrations of NaCl and KCl added to the sample and (B) peak efficiencies of ␥-globulin at different concentration of NaCl and NaI added to the sample analyzed on M7C4I coated capillaries. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3; approximately 50 nL of the sample was injected. The data is the average of triplicate injections with the CV for efficiency ranging from 0.02 to 2.4% for transferrin, and 0.01 to 2.4% for ␥-globulin.
3.2. Effect of different alkali salts on the peak efficiency using M7C4I coatings Other alkali salts, potassium chloride (KCl) and sodium iodide (NaI), were added to the protein samples at identical conditions as for NaCl to evaluate if similar results could be obtained as for NaCl. The effect of different concentrations of the added salts (0–100 mM) was studied for the proteins transferrin and ␥-globulin. The results indicate that KCl has an improving effect on the peak efficiency of transferrin, however, not as strong as NaCl (Fig. 4A) (CV for efficiency ranging from 0.02 to 1.4% for NaCl and 0.03 to 2.4 for KCl based on triplicate injections). Furthermore, when KCl was added to 50–70 mM concentration to the sample, there was a sudden drop in the peak efficiency for transferrin. The obtained results indicate that the addition of alkali salts to the sample has a positive effect on the peak efficiency and that the effect of Na+ seems to be more pronounced than K+ . The effect of using another halogen than chloride was investigated by adding different concentrations of NaI to samples of ␥-globulin, see Fig. 4B. The obtained results showed that the peak efficiency can also be improved by NaI salt addition. However, the shape of the peak efficiency curves differs. At low salt concentration (up to 40 mM) somewhat lower efficiencies were obtained
Mixtures of four proteins (lysozyme, BSA, transferrin and ␥globulin) in equal molar concentrations (1.5 M) were dissolved in different concentrations of NaCl and analyzed to study the effect of salt additions on the resolution of the separation. The resolution in the separation increased when low molar concentrations of NaCl were added to the sample compared to separation of proteins dissolved in pure MQ-water (Fig. 5). However, as the added salt concentration exceeds 15–20 mM NaCl, the proteins mixture start to co-migrate into an even sharper peak. At higher concentration than 20 mM, no separation of the proteins could be observed. A possible explanation to the co-migration at high salt concentrations could be that all proteins, regardless of their mass, will reach the same, or very similar charge-to-size ratio due to the excess of salt and thus the same electrophoretic migration. To evaluate this, the migration times for the individually injected proteins was normalized against the EOF (calculated by the water dip in the electropherograms) and plotted in Fig. 6. The normalized values all reach the same value above 40 mM NaCl, except for lysozyme. This supports the hypothesis of protein charging to reach the same charge-to-size ratio, regardless of MW of the protein. 3.4. Effect of salt concentration on the peak efficiency using PolyE-323 surfaces To investigate if the effect of the improvement of the efficiency in the separation when adding salt to the sample was surface coating dependent, other coatings were investigated. The PolyE-323 [23,25] polymer interacts electrostatically with the ionized silanol groups and as it is a polycationic polymer it yields a positive surface with reversed EOF, similar to the M7C4I coated silica capillaries. This coating has previously been developed by our group and has shown to be suitable for bioanalytical applications [23,25]. The analysis of transferrin at different NaCl concentrations (0–100 mM) is shown in Fig. 7. The peak intensity and efficiencies were much lower compared to the covalent M7C4I coating and no peak stacking effects could be observed with increasing salt concentration. One possible explanation could be that presence of salt reduces the electrostatic interactions between PolyE-323 and the silica wall. However, that would have reduced the EOF at higher salt concentrations, which was not observed. 3.5. Effect of salt concentration on the peak efficiency using TAC surfaces Another covalent monomer coating was tested to evaluate if the M7C4I results could be obtained with a different positively charged monomer coating. For this purpose, analyses were carried out using the TAC monomer for silica surface modification. The TAC coating attaches covalently to the silanol groups, producing a stable, positively charged surface and reversed EOF [19]. The
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Fig. 6. Normalized migration times for the proteins lysozyme, ␣-chymotrypsinogen transferrin and ␥-globulin as a function of salt concentration added to the protein sample. The migration times were normalized against the water dip in the electropherrograms. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more.
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Fig. 8. CE–UV analysis of transferrin on TAC coated capillary as a function of the NaCl concentration (0–100 mM) in the protein sample. The transferrin concentration was 1.5 M. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more.
bic properties than M7C4I, which could also partly explain the results. 3.6. Effect of salt concentration on the peak efficiency using neutral PVA surfaces
Fig. 7. CE–UV analysis of transferrin by adding different concentrations of NaCl (0–100 mM) to the sample on PolyE-323 coated capillaries. The protein concentration was 1.5 M. Conditions: capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection, 1.5 M protein solution was injected at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3. All injections were carried out in triplicates or more.
influence of adding different NaCl concentrations in a transferrin sample was studied. The highest peak efficiency for transferrin was obtained at a much lower salt concentration (10 mM) compared to what was needed to get the maximum efficiency with the M7C4I coating (50 mM) as shown in Fig. 8. A possible explanation for the lower salt concentration needed for optimal efficiencies on the TAC coating could be related to a higher charge density obtained with this coating due to the smaller molecular size of the coating reagent. This assumption is supported by the observed higher EOF obtained for the TAC coating compared to the M7C4I. Furthermore, the TAC coating reagent has less hydropho-
The M7C4I, PolyE-323 and TAC coatings all generate a positively charged surface with high electroosmotic flows. The positive surface for these coatings will have electrostatic repulsion effects on the proteins, especially when a low pH is used in the BGE and sample plug and when an alkali salt is added to the sample. PVA coated capillaries have a hydrophilic and neutral surface, thus generating no EOF in CE analysis. These capillaries are commercially available and readily used for protein and peptide separation with CE [37,38]. Therefore, PVA coated capillaries were tested to investigate if the observed peak stacking effect could be obtained with a neutral surface. Transferrin dissolved in Milli-Q water and different NaCl concentrations (0–100 mM) or dissolved in BGE (10 mM ammonium formate pH 3.0) and different NaCl concentrations (0–100 mM) were studied. The peak efficiencies and peak heights increased (9.2 × 103 plates/m to 1.7 × 106 plates/m and 21 mAU to 440 mAU respectively for Milli-Q–NaCl and 8.7 × 103 plates/m to 1.1 × 106 plates/m and 17 mAU to 413 mAU respectively for BGE–NaCl) with increasing NaCl concentrations (data not shown). A difference to the positively charged surfaces was that the highest peak efficiencies were reached at the highest NaCl-concentration tested, i.e., no maximum value as observed for the other coatings was obtained. Interestingly, no difference between dissolving transferrin in MQ–NaCl or BGE–NaCl could be observed, which is consistent with the results for the M7C4I coating and showing that the effect is obtained also with a proper pH-control of the introduced sample. 4. Conclusions The effect of adding different concentrations of alkali salts to protein samples with a large range of molecular weight
Fig. 5. CE–UV separations of a mixture of the proteins lysozyme, BSA, transferrin and ␥-globulin on M7C4I coated capillary as a function of the NaCl concentration added to the protein sample. The concentration of each protein in the sample was 1.5 M. Other conditions; capillaries, 50 m I.D., 365 m O.D., 50 cm total length and 41.5 cm to the detection window; injection at 50 mbar for 30 s; applied negative voltage, −25 kV; detection at 200 nm; BGE, 10 mM ammonium formate, pH 3; approximately (A) MQ water, (B) 3 mM NaCl, (C) 5 mM NaCl, (D) 10 mM NaCl, (E) 15 mM NaCl, (F) 20 mM NaCl, (G) 30 mM NaCl and (H) 70 mM NaCl.
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(14–669 kDa) analyzed by CE with various surface modified capillaries was conducted in this study. Regardless of molecular weight, a dramatic improvement in peak efficiency and peak height was observed when adding the alkali salts to the protein sample except for the PolyE-323 polymer coated capillaries. This coating is not covalently bound to the silica surface. It is electrostatically attracted to the silanols, which is a major difference compared to the other surfaces investigated. The optimal concentration of alkali salts needed in the sample plug is dependent on protein MW and molar concentration of the protein, indicating a protein–salt interaction phenomenon. In addition, the optimal salt concentration varies depending on the surface coating used implying that the results also are due to salt–surface interactions. These interactions will differ depending on the nature of the surface and thus explain the observed differences between the different coatings. Therefore, based on the obtained results with the M7C4I coating, in combination with PolyE-323, TAC and PVA, it is highly likely that the observed peak sharpening effect when adding salt to the sample is a mixed effect of both protein–salt and salt–surface interactions. Adding salts to a protein mixture will initially increase the peak efficiencies and resolution. However, an excess of salt will cause all proteins in the mixture to co-migrate in one sharp peak and thereby ruin the separation. A possible explanation for this is that the proteins will reach very similar charge-to-size ratios and therefore not separated in the CE analysis. In this study, we are reporting the interesting and somewhat surprising peak stacking effect for proteins when alkali salts were added to the sample. The underlying mechanism behind the observed effect is still unclear. Therefore, further studies regarding this phenomenon are planned and will be conducted. Acknowledgments This work was supported by a PhD grant from the Libyan government to Anisa Elhamili. A. Puerta acknowledges Spanish Ministry of Education and Science for a postdoctoral grant to perform research in Sweden. The Swedish Research Council is acknowledged for their support (grants, 629-2005-5379 and 629-2002-6821). The authors would like to thank Prof. Roberto Sebastiano and Prof. Pier Giorgio Righetti for supplying the M7C4I coating, and we would also like to acknowledge Prof. Jan Ståhlberg for additional fruitful discussions regarding the obtained results.
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