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Qingfu Zhu1 Stefan H. Heinemann2 2 ¨ Roland Schonherr Gerhard K. E. Scriba1 1 Department
of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller University Jena, Jena, Germany 2 Department of Biophysics and Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany Received August 1, 2014 Revised September 3, 2014 Accepted September 4, 2014
Research Article
Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual † cyclodextrin-crown ether systems A dual-selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine R 21 and sulfoxide. The combinations of the crown ethers 15-crown-5, 18-crown-6, Kryptofix R Kryptofix 22 and -cyclodextrin, carboxymethyl--cyclodextrin, and sulfated -cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 m id fused-silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated -cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the R diaza-crown applied cyclodextrin, the addition of crown ethers, especially the Krpytofix ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide-crown ether-cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system. Keywords: Capillary electrophoresis / Crown ethers / Cyclodextrin / Methionine sulfoxide / Peptide diastereomers DOI 10.1002/jssc.201400825
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction L-Methionine
(Met) residues in peptides and proteins are susceptible to oxidation to methionine sulfoxide [Met(O)] by reactive oxygen species generated in physiological reactions and under oxidative stress conditions [1]. Oxidized peptides accumulate during aging [2] and are thought to play a role in degenerative diseases such as morbus Alzheimer [3]. Protein-bound as well as free Met(O) can be reduced by methionine sulfoxide reductase (Msr) enzymes, a group of thiol oxidoreductases which protect cells against oxidative damage [4]. Because of the chirality of the sulfoxide moiety, Met(O) exists as a pair of diastereomers, i.e.,
Correspondence: Professor Gerhard K. E. Scriba, Department of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller University Jena, Philosophenweg 14, 07743 Jena, Germany E-mail: [email protected] Fax: +49-3641-949802
Abbreviations: Ac, acetyl; CM--CD, carboxymethyl-cyclodextrin; dabsyl, 4-N,N-dimethylaminoazobenzene-4 sulfonyl; Dnp, 2,4-dinitrophenyl; Met(O), L-Methionine sulfoxide; Met-S-(O), L-methionine-(S)-sulfoxide; Msr, methionine sulfoxide reductase C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
L-methionine-(S)-sulfoxide [Met-S-(O)] and L-methionine-(R)sulfoxide [Met-R-(O)]. Consequently, two types of Msr enzymes exist which reduce Met(O) in a stereospecific manner. Reduction of Met-S-(O) is catalyzed by MsrA, while reduction of the R-configured sulfoxide is mediated by MsrB [5, 6]. To study the stereospecificity of Met(O) reduction by Msr enzymes, the individual diastereomers 4-N,N-dimethylaminoazobenzene-4 -sulfonyl (dabsyl)-Met-S(O) and dabsyl-L-methionine-(R)-sulfoxide have been frequently used as substrates [7–9]. Furthermore, CE-based assays separating the diastereomers of dabsyl-Met(O) [10], 9-fluorenylmethyloxycarbonyl (Fmoc)-Met(O) [11] or the Nacetylated, C-terminal 2,4-dinitrophenyl (Dnp)-labeled peptide Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp [12] have been reported. CE has matured to one of the major separation technique for peptide analysis [13–15] including the separation of peptide enantiomers and diastereomers [16–19]. Furthermore, the successful separation of aryl and alkyl sulfoxide enantiomers [20, 21] as well as the sulfoxide enantiomers of the drugs albendazole [22] and thioridazine [23] has
† This paper is included in the virtual special issue sample preparatioin mass spectrometry available at the Journal of Separation Science website.
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been accomplished. Recently, the first separation of peptide diastereomers containing Met(O) has been described by our laboratory [12]. The diastereomers Ac-Lys-Ile-Phe-Met(O)Lys-Dnp were resolved by the use of a dual selector system composed of sulfated -CD and the achiral crown ether 15crown-5. Therefore, the aim of the present study was a further investigation of the dual selector system for Met(O) peptide diastereomer separation including several CDs as well as achiral crown ethers. Synthetic N-acetylated pentapeptides containing a Dnp label at the C terminus were studied.
2 Materials and methods 2.1 Chemicals The Met(O) peptide diastereomers, Ac-Lys-Asp-Met(O)Asp-Lys-Dnp, Ac-Lys-Asp-Met(O)-Asn-Lys-Dnp, Ac-LysAsn-Met(O)-Asp-Lys-Dnp, Ac-Lys-Val-Met(O)-Val-Lys-Dnp, Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp and Ac-Lys-Ile-Phe-Met(O)Lys-Dnp, were purchased from Peptide Specialty Laboratories (Heidelberg, Germany). -Cyclodextrin (-CD), ␥-CD, methyl--CD, 2-hydroxypropyl--CD, heptakis(2,6-diO-methyl)--CD, sulfopropyl--CD, sulfobutyl ether--CD, carboxymethyl-␥-CD and carboxymethyl--CD (CM--CD) were obtained from Cyclolab (Budapest, Hungary), while sulfated -CD, sulfated ␥-CD and Tris were from SigmaAldrich (Deisenhofen, Germany). Citric acid monohydrate, R 21 (7,13-diaza-1515-crown-5, 18-crown-6, Kryptofix crown-5, 1,4,10-trioxa-7,13-diazacyclopentadecane) and R 22 (4,13-diaza-18-crown-6, 1,7,10,16-tetraoxaKryptofix 4,13-diazacyclooctadecane) were purchased from MerckMillipore (Darmstadt, Germany). HPLC-grade acetonitrile and methanol were from VWR International (Darmstadt, Germany). All other chemicals were of the highest purity commercially available and used without further purification. Water was purified by a Milli-Q Direct 8 system (Merck-Millipore, Darmstadt, Germany).
the pH of 50 mM phosphoric acid with 1 M sodium hydroxide solution. All buffers were filtered (0.2 m) and sonicated for 5 min before use. 2 mM stock solutions of the peptides were prepared in water. Working solutions were obtained by a tenfold dilution with water before the injection. 2.3 Instrumentation All CE experiments were performed using a Beckman P/ACE MDQ CE system (Beckman Coulter, Krefeld, Germany) equipped with a UV-Vis diode array detector and a sample tray temperature control system set at 10⬚C. Instrument control and data evaluation were accomplished by the 32 KaratTM Software. A 40/50.2 cm, 50 m id, 363 m od fused-silica capillary (BGB Analytik Vertrieb, Schlossb¨ockelheim, Germany) was used. A new capillary was successively rinsed at a pressure of 138 kPa (20 psi) with 1 M sodium hydroxide for 20 min, water for 10 min, 0.1 M sodium hydroxide for 20 min, water for 10 min, and the BGE for 10 min. At the end of each day, the capillary was rinsed with 0.1 M sodium hydroxide for 10 min, water for 10 min and left in water overnight. Between analyses, the capillary was flushed with 0.1 M sodium hydroxide for 3 min, water for 2 min, and the BGE for 3 min. The separation voltage was 25 kV and the capillary temperature was set at 20⬚C. Hydrodynamic sample injection was performed at a pressure of 3.4 kPa (0.5 psi) for 4 s. UV detection was carried out at 214 nm either at the cathodic end or at the anodic end upon reversal of the separation voltage. 2.4 Calculations Peptide pI values were calculated using the GenScript Peptide Property Calculator (www.genscript.com/sslbin/site2/peptide_calculation.cgi). Peak resolution was calculated by the 32 KaratTM Software according to RS = 2(t2 – t1 )/(w1 +w2 ) where t1 and t2 are the migration times, and w1 and w2 are the widths of the peaks at the baseline.
2.2 BGEs and solutions
3 Results and discussion 50 mM Tris-citric acid buffers were prepared by dissolving the appropriate amount of Tris in water and adjusting the pH with 1 M citric acid before completing to the desired volume with water. For buffers containing a single selector, the appropriate amount of CD or crown ether was added to 25 mL of the respective Tris-citric acid buffer. After addition R diaza-crown ethers, the pH of the buffer of the Kryptofix was readjusted using a 1 M solution of citric acid. For the buffers containing two selectors, the appropriate amount of the crown ether was first added to 50 mL of the Tris-citric acid buffer. As in the case of single selector-containing buffers, the pH of the buffer was readjusted using a 1 M solution R diaza-crown of citric acid after the addition of Kryptofix ethers. Subsequently, the appropriate amount of a CD was added to 25 mL of the crown ether-containing Tris buffer. A 50 mM sodium phosphate buffer was prepared by adjusting C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.1 Screening of CDs for diastereomer separation Evaluation of the separation conditions was performed using the N-acetylated and C-terminal Dnp-labeled pentapeptides Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp, Ac-LysAsn-Met(O)-Asp-Lys-Dnp, Ac-Lys-Val-Met(O)-Val-Lys-Dnp, Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp and Ac-Lys-Ile-Phe-Met(O)Lys-Dnp. Assuming that the oxidation of Met does not affect the pI of peptides, Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp can be considered a neutral peptide with a pI of about 7.0. A pI value of 8.9 was calculated for Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp, while the values of the remaining peptides were 10.5 (Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp) and 10.8 (Ac-Lys-ValMet(O)-Val-Lys-Dnp and Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp). Thus, all peptides are positively charged in the acidic pH www.jss-journal.com
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range and, except for Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp, also in the slight alkaline pH range. Therefore, the separation was investigated in a fused-silica capillary in the pH range 2.5–9.5 using 50 mM Tris-citric acid buffers or phosphate-based buffers. However, no separation of the peptide diastereomers was observed under these conditions. Subsequently, neutral and charged CDs including native -CD and ␥-CD, methyl--CD, 2-hydroxypropyl-CD, heptakis(2,6-di-O-methyl)--CD, sulfopropyl--CD, sulfobutyl ether--CD, sulfated -CD, sulfated ␥-CD, carboxymethyl--CD (CM--CD) and carboxymethyl-␥-CD were evaluated as buffer additives at a concentration of 15 mg/mL in a 50 mM Tris-citric acid buffer at pH 2.5 and pH 8.0. At pH 2.5, only a very limited partial separation was observed for Ac-Lys-Ile-Phe-Met(O)-LysDnp and Ac-Lys-Val-Met(O)-Val-Lys-Dnp in the presence of CM--CD. None of the analytes could be detected within 100 min under normal polarity of the separation voltage in the presence of sulfated -CD even when reducing the concentration of the selector to 2 mg/mL. Consequently, the concentration of sulfated -CD was raised to 30 mg/mL and the polarity of the separation voltage was reversed. Under these conditions, Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp and Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp could be detected within 10 min but no peptide diastereomer separation was observed. At pH 8.0, partial separation of the diastereomers of Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp, Ac-Lys-Glu-Met(O)-LysLys-Dnp, Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp and Ac-Lys-AsnMet(O)-Asp-Lys-Dnp was noted in the presence of 15 mg/mL sulfated -CD with normal polarity of the separation voltage. Baseline separation (Rs > 1.5) of Ac-Lys-Ile-Phe-Met(O)-LysDnp and Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp was achieved in a BGE containing 20 mg/mL sulfated -CD, 50 mM Tris-citric acid buffer, pH 7.5. The other peptide diastereomers could not be baseline resolved no matter what sulfated -CD concentration or buffer pH was investigated. All other investigated CDs proved to be ineffective as BGE additives for achieving at least a partial separation under the experimental screening conditions.
3.2 Screening of the dual system of achiral crown ethers and CDs It has been reported in the literature that the addition of achiral crown ethers can enhance enantioseparations of primary amines including amino acids. 18-crown-6 [24–26] or diaza-crown ethers [27, 28] have been used in combination with native -CD or neutral -CD derivatives. These enantioseparations are assumed to be based on the formation of a sandwich-type complex between the analyte enantiomers and the respective CDs and crown ethers [29, 30]. Consequently, the crown ethers 18-crown-6 and 15-crown-5 as well R 22 (4,13-diaza-18-crownas the diaza-crown ethers Kryptofix R 6) and Krpytofix 21 (7,13-diaza-15-crown-5) were studied in combination with -CD, CM--CD and sulfated -CD using Tris-citric acid buffers with a pH of 2.5 and 8.0 as BGEs. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The concentration of the CDs was set at 10 mg/mL, while 18-crown-6 and 15-crown-5 were tested at a concentration of R 15 mM and the nitrogen-containing Krpytofix crown ethers at 8 mM. The results in comparison with the sole use of CDs as additives are summarized in Supporting Information Tables S1 (pH 2.5) and S2 (pH 8.0). It should be noted that addition of a crown ether to the BGE in the absence of a CD in concentrations up to 20 mM did not result in any separation. The neutral crown ethers, 18-crown-6 and 15-crown-5, displayed a synergistic effect on the diastereomer separation in combination with CM--CD at pH 2.5 and with sulfated -CD at pH 8.0. While partial separations were observed at pH 2.5, baseline separations could be observed for AcLys-Ile-Phe-Met(O)-Lys-Dnp and Ac-Lys-Glu-Met(O)-Lys-LysDnp with sulfated -CD and the crown ethers. Migration times increased in the presence of the crown ethers compared to the sole presence of CDs indicating that a ternary complex may be formed between peptide diastereomers, CDs and crown ethers as derived for amine analytes [24, 29, 30]. No separation could be observed in combination with native -CD even at concentrations of up to 25 mg/mL of the CD. The pKa values of diaza-crown ethers are about 9 [31], so that the compounds are protonated at the pH values studied. Interestingly, none of the analytes could be detected at R 22 under both, norpH 2.5 in the presence of Kryptofix mal and reversed polarity of the separation voltage. A similar behavior has also been observed by Iv´anyi and colleagues when attempting the enantioseparation of Trp by the comR 22 and neutral CD derivatives [28]. bination of Kryptofix In contrast, partial diastereomer separation of several pepR 21. It may be speculated tides were observed for Kryptofix R that the protonated Kryptofix 22 is adsorbed to the capillary wall, thus, modifying the EOF, while this occurs to a R 21 crown ether. lesser extent in case of the smaller Kryptofix Diaza-crown ethers have been used for EOF modification and reversal for the separation of inorganic ions as bifunctional modifiers [32, 33]. Migration times increased as compared to the presence of the neutral crown ethers despite the fact that the charge of the complexes should be higher in the case of the protonated diaza-crown ethers. This further indicates that the diaza-crown ethers induced an EOF directed toward the anode. Finally, the diastereomers of Ac-Lys-Ile-Phe-Met(O)R 21 Lys-Dnp were resolved by the combination of Kraptofix and -CD at pH 2.5. A significant synergistic effect between the CDs and the diaza-crown ethers was noted at pH 8.0. Except for Ac-LysAsp-Met(O)-Asp-Lys-Dnp with the approximate pI of 7.0, all other peptide diastereomers were at least partially separated using CM--CD. Effective separations were found in the presence of sulfated -CD with baseline resolution RS > 5 for Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp in the presence of both diaza-crown ethers. The diastereomers of this peptide were even resolved when -CD was used as CD component. Figure 1 illustrates the effect of the crown ethers on the diastereomer separation of Ac-Lys-Glu-Met(O)-LysLys-Dnp under the screening conditions at pH 8.0 as an example. www.jss-journal.com
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J. Sep. Sci. 2014, 00, 1–7 Figure 1. Effect of CDs and crown ethers as BGE additives on the separation of the diastereomers of Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp. Experimental conditions: 40/50.2 cm, 50 m id fused-silica capillary, 50 mM Tris-citric acid buffer, pH 8.0, 25 kV, 20⬚C, detection at 214 nm. Buffer additives (A) 10 mg/mL sulfated -CD, (B) 10 mg/mL sulfated -CD and 15 mM 15-crown-5, (C) 10 mg/mL sulfated -CD and 15 mM 18-crown-6, (D) 10 mg/mL R sulfated -CD and 8 mM Kryptofix 21, and (E) R 10 mg/mL sulfated -CD and 8 mM Kryptofix 22.
3.3 Effect of CD and crown ether concentration in the dual system The effect of different CD and crown ether concentrations on the separation of the peptide diastereomers was studied R 22 as well as sulfated using 18-crown-6 and Kryptofix CD, except for Ac-Lys-Val-Met(O)-Val-Lys-Dnp. In this case, CM--CD was applied because it proved to be an effective additive for the diastereomer separation of this peptide in the screening experiments. The experiments were conducted at pH 8.0 because efficient separations were observed at this pH in contrast to pH 2.5. The concentration of one type of selector was fixed while the concentration of the second selector was varied. The results are summarized in Fig. 2. Generally, with a few exceptions, an increase of peak resolution and migration time can be observed upon increasing the concentration of a selector while keeping the other selector constant. In the case of Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp an optimum appears to exist for a CD concentration (with constant crown ether concentrations) of about 15 mg/mL because peak resolution decreased when further increasing the CD concentration (Fig. 2B and D). Only partial resolution of the diastereomers of Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp was R 22 < 10 mM, while observed at concentrations of Kryptofix no separation was observed at higher concentrations. In contrast to the other systems, migration analyte times R 22-based BGE upon addecreased in case of the Kryptofix dition of 5 mg/mL of the CDs and subsequently increased at higher CD concentrations (Fig. 2H). This decrease did not correlate with a general decrease in peak resolution (Fig. 2D). As stated above, it may be speculated that the positively charged diaza-crown ether is adsorbed to the capillary wall resulting in a slow EOF directed toward the anode. This can be counteracted by the presence of negatively charged CDs such as sulfated -CD or CM--CD resulting in a faster migration of the positively charged analytes to the detector at the cathodic end of the capillary. The subsequent increase of the migration times at higher CD concentrations can be explained by the increased analyte complexation and increased viscosity of the buffer at higher CD concentrations.
3.4 Effect of buffer pH The effect of the pH of the Tris-citric acid-based electrolyte was studied in the pH range 2.5 to 9.5 in BGEs C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
containing 15 mg/mL CM--CD and 20 mM 18-crown-6 or R 22. The diaza-crown ether is only partially 10 mM Kryptofix protonated above pH 9.0, while CM--CD will be protonated at pH < 3–4. Ac-Lys-Val-Met(O)-Val-Lys-Dnp was used as model peptide because separations in CM--CD-based BGEs have been observed during the screening experiments. The peptide is positively charged over the entire pH range studied (pI of approximately 10.8). Increasing the pH of the BGE containing CM--CD and 18-crown-6 resulted in a deterioration of the diastereomer separation with a concomitant decrease of migration times (data not shown). No separation could be observed at pH values above 4. This may be explained by the increased EOF at higher pH values leading to short migration times, which did not allow sufficient time for analyte-selector interactions. R 22, long For the combination CM--CD and Kryptofix migration times were observed at pH values < 6.5. The dependence of the resolution of the diastereomers of Ac-LysVal-Met(O)-Val-Lys-Dnp in the pH range 6.5–9.5 is depicted in Fig. 3. The highest resolution was found at pH 7.5 to 8. Further increase of the pH resulted in a loss of resolution. Migration times also significantly decreased, e.g. from about 45 min at pH 7.5 to about 25 min at pH 8.5 with a concomitant decrease of resolution from 2.4 to 2.1. Thus, the recommended pH for the separation would be pH 8.0 to 8.5 as a compromise of resolution and analysis time. It may be speculated that the decreasing migration times (and loss of R 22 is less proresolution) are due to the fact that Kryptofix tonated at pH 8–8.5 so that interaction of crown ether with the capillary wall is weakened resulting in a change of the EOF directed toward the cathode. The effect of the change of the charge of the selectors as a function of buffer pH on the formation of the ternary complex with the positively charged peptide cannot be concluded from the present data.
3.5 Diastereomer separation of a peptide mixture The simultaneous separation of the diastereomers of Ac-LysAsp-Met(O)-Asp-Lys-Dnp, Ac-Lys-Asp-Met(O)-Asn-Lys-Dnp and Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp was also studied. The peptides differ in an exchange of the acidic amino acid Asp with the neutral amino acid Asn either N- or C-terminal to Met(O), which results in a basic pI value of about 8.9 versus the pI of about 7.0 of the peptide containing two Asp residues. The electropherograms are shown in Fig. 4. In the www.jss-journal.com
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Figure 2. Effect of the concentration of CDs and R the crown ethers, 18-crown-6 and Kryptofix 22, on (A–D) resolution of Met(O) peptide diastereomers and (E–H) migration time of the first migrating diastereomer. Experimental conditions: 40/50.2 cm, 50 m id fusedsilica capillary, 50 mM Tris-citric acid buffer, pH 8.0, 25 kV, 20⬚C, detection at 214 nm. CM-CD was used for the separation of Ac-Lys-ValMet(O)-Val-Lys-Dnp while sulfated -CD was used for the separation of the other peptides. The concentration of the CD was fixed at 10 mg/mL when varying the crown ether concentrations (A, C, E, G). The concentrations of 18-crown-6 was fixed at 15 mM (B, R F) and Kryptofix 22 at 8 mM (D, H) when varying the CD concentration. 䊉, Ac-Lys-AspMet(O)-Asp-Lys-Dnp; 䉱, Ac-Lys-Val-Met(O)-ValLys-Dnp; 䊊, Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp; , Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp; , Ac-LysIle-Phe-Met(O)-Lys-Dnp.
Figure 3. pH-Dependence of the separation of the Ac-LysVal-Met(O)-Val-Lys-Dnp diastereomers. Experimental conditions: 40/50.2 cm, 50 m id fused-silica capillary, 50 mM Tris-citric acid R buffer, 15 mg/mL CM--CD, 10 mM Kryptofix 22, 25 kV, 20⬚C, detection at 214 nm.
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absence of CDs and/or crown ethers, the three peptides migrated as a single peak. The peptides were separated in the presence of 12 mg/mL sulfated -CD but only partial diastereomer separation of the Asn-containing peptides were observed (Fig. 4A). As expected, improvement of the diastereomer resolution could be achieved by further addition of the R 22 (Fig. 4C). crown ethers, 18-crown-6 (Fig. 4B) or Kryptofix However, the separation of the diastereomers of Ac-Lys-AspMet(O)-Asp-Lys-Dnp was still incomplete. Thus, addition of organic solvents was considered which may affect the interactions between analytes and BGE additives. Subsequently, 15% v/v of acetonitrile or methanol were added to the BGE. While the presence of methanol did not affect the diastereomer separation and significantly prolonged migration times,
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been combined with the crown ethers. The present data on peptide diastereomer separations indicate that crown ethers can also be effectively combined with negatively charged CDs for stereoisomer separations. The financial support of Q. Zhu by the China Scholarship Council (CSC) is gratefully acknowledged. The authors have declared no conflict of interest.
5 References [1] Voigt, W., Free Radic. Biol. Med. 1995, 18, 93–105. [2] Stadtman, E. R., Free Radic. Res. 2006, 40, 1250–1258. ¨ [3] Schoneich, C., Biochim. Biophys. Acta 2005, 1703, 111–119. [4] Moskovitz, J., Bar-Noy, S., Williams, W. M., Requena, J., Berlett, B. S., Stadtman, E. R., Proc. Natl. Acad. Sci. USA 2001, 98, 12920–12925. [5] Kim, H. Y., Gladyshev, V. N., Biochem. J. 2007, 407, 321–329. [6] Lee, B. C., Gladyshev, V. N., Free Radic. Biol. Med. 2011, 50, 221–227.
Figure 4. Electropherograms of separation of the peptide diastereomers Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp, Ac-Lys-AspMet(O)-Asn-Lys-Dnp, and Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp. Experimental conditions: 40/50.2 cm, 50 m id fused-silica capillary, 50 mM Tris buffer, pH 8.0, 25 kV, 20⬚C, detection at 214 nm. Buffer additives are (A) 12 mg/mL sulfated -CD, (B) 12 mg/mL sulfated -CD plus 25 mM 18C6, (C) 12 mg/mL sulfated -CD plus 6 mM R Kryptofix 22, (D) 12 mg/mL sulfated -CD, 25 mM 18-crown-6, plus 15% acetonitrile.
addition of acetonitrile significantly improved the resolution of Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp as shown in Fig. 4D for the combination of sulfated -CD and 18-crown-6.
[7] Moskovitz, J., Poston, J. M., Berlett, B. S., Nosworthy, N. J., Szczepanowski, R., Stadtman, E. R., J. Biol. Chem. 2000, 275, 14167–14172. [8] Minetti, G., Balduini, C., Brovelli, A., Ital. J. Biochem. 1994, 43, 273–283. [9] Uthus, E. O., Anal. Biochem. 2010, 401, 68–73. ¨ [10] Zhu, Q., El-Mergawy, R. G., Heinemann, S. H., Schonherr, ˇ P., Scriba, G. K. E., Electrophoresis 2013, 34, R., Ja´ c, 2712–2717. ¨ [11] Zhu, Q., El-Mergawy, R. G., Heinemann, S. H., Schonherr, ˇ P., Scriba, G. K. E., Anal. Bioanal. Chem. 2014, R., Ja´ c, 406, 1723–1729. ¨ [12] Zhu, Q., Huo, X., Heinemann, S. H., Schonherr, R., ElMergawy, R., Scriba, G. K. E., J. Chromatogr. A 2014, 1359, 224–229. ˇ cka, ˇ [13] Kasi V., Electrophoresis 2010, 31, 122–146.
4 Concluding remarks
ˇ cka, ˇ [14] Kasi V., Electrophoresis 2012, 33, 48–73. ˇ cka, ˇ [15] Kasi V., Electrophoresis 2014, 35, 69–95.
A dual selector system has been developed for the separation of the diastereomers of Met(O)-containing peptides by CE. Combinations of the crown ethers 18-crown-6 and 15-crown-5 R R 21, Kryptofix 22 as well as the diaza-crown ethers Kryptofix and -CD derivatives were evaluated. Especially dual systems containing sulfated -CD or CM--CD and a diaza-crown ether could be used for the resolution of the Met(O) peptide diastereomers in pH 8.0 BGEs. This is interesting to note because both, peptide and diaza-crown ether, are at least partially positively charged at this pH. The present data further support the additive effect of CDs and crown ethers for aminecontaining analytes as reported for enantiomer separations in the literature [24–30]. In these studies, neutral CDs have C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[16] Czerwenka, C., Lindner, W., Anal. Bioanal. Chem. 2005, 382, 599–638. [17] Scriba, G. K. E., Electrophoresis 2006, 27, 222–230. [18] Scriba, G. K. E., Electrophoresis 2009, 30, S222–S228. [19] Ali, I., Al-Othman, Z. A., Al-Warthan, A., Asnin, L., Chudinov, A., J. Sep. Sci. 2014, 37, 2447–2466. [20] Hadley, M. R., Decrette, M., Guillore, G., Rosini, C., Donnoli, M. I., Superchi, S., Hutt, A. J., J. Sep. Sci. 2001, 24, 766–776. [21] Rodriguez, M. A., Liu, Y., McCulla, R., Jenks, W. S., Armstrong, D. W., Electrophoresis 2002, 23, 1561–1570. [22] Prost, F., Caslavska, J., Thormann, W., J. Sep. Sci. 2002, 25, 1043–1054.
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[23] Masetto de Gaitani, C., Martinez, A. S., Bonato, P. S., Electrophoresis 2003, 24, 2723–2730.
´ ´ K., Laz ´ ar, ´ I., Massart, D. L., Vander Heyden, [28] Ivanyi, T., Pal, Y., J. Chromatogr. A 2004, 1028, 325–332.
[24] Huang, W. X., Fazio, S. D., Vivilecchia, R. V., J. Chromatogr. A 1997, 781, 129–137.
[29] Zhang, C., Huang, W. X., Chen, Z., Rustum, A. M., J. Chromatogr. A 2010, 1217, 4965–4970.
[25] Armstrong, D. W., Chang, L. W., Chang, S. S. C., J. Chromatogr. A 1998, 793, 115–134.
[30] Koide, T., Ueno, K., J. Chromatogr. A 2001, 923, 229–239.
[26] Elbashir, A. A., Suliman, F. O., J. Chromatogr. A 2011, 1218, 5344–5351 ´ ´ ar, ´ I., Vander Hey[27] Elek, J., Mangelings, D., Ivanyi, T., Laz den, Y., J. Pharm. Biomed. Anal. 2005, 38, 601–608.
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[31] Dantz, D. A., Buschmann, H.-J., Schollmeyer, E., Thermochim. Acta 1997, 294, 133–138. [32] Chiou, C.-S., Shih, J. S., Analyst 1996, 121, 1107–1110. [33] Mori, M., Tsue, H., Tanaka, S., Tanaka, K., Haddad, P., Electrophoresis 2003, 24, 1944–1950.
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Qingfu Zhu1 Stefan H. Heinemann2 2 ¨ Roland Schonherr Gerhard K. E. Scriba1 1 Department
of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller University Jena, Jena, Germany 2 Department of Biophysics and Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany Received August 1, 2014 Revised September 3, 2014 Accepted September 4, 2014
Research Article
Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual † cyclodextrin-crown ether systems A dual-selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine R 21 and sulfoxide. The combinations of the crown ethers 15-crown-5, 18-crown-6, Kryptofix R Kryptofix 22 and -cyclodextrin, carboxymethyl--cyclodextrin, and sulfated -cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 m id fused-silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated -cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the R diaza-crown applied cyclodextrin, the addition of crown ethers, especially the Krpytofix ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide-crown ether-cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system. Keywords: Capillary electrophoresis / Crown ethers / Cyclodextrin / Methionine sulfoxide / Peptide diastereomers DOI 10.1002/jssc.201400825
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction L-Methionine
(Met) residues in peptides and proteins are susceptible to oxidation to methionine sulfoxide [Met(O)] by reactive oxygen species generated in physiological reactions and under oxidative stress conditions [1]. Oxidized peptides accumulate during aging [2] and are thought to play a role in degenerative diseases such as morbus Alzheimer [3]. Protein-bound as well as free Met(O) can be reduced by methionine sulfoxide reductase (Msr) enzymes, a group of thiol oxidoreductases which protect cells against oxidative damage [4]. Because of the chirality of the sulfoxide moiety, Met(O) exists as a pair of diastereomers, i.e.,
Correspondence: Professor Gerhard K. E. Scriba, Department of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller University Jena, Philosophenweg 14, 07743 Jena, Germany E-mail: [email protected] Fax: +49-3641-949802
Abbreviations: Ac, acetyl; CM--CD, carboxymethyl-cyclodextrin; dabsyl, 4-N,N-dimethylaminoazobenzene-4 sulfonyl; Dnp, 2,4-dinitrophenyl; Met(O), L-Methionine sulfoxide; Met-S-(O), L-methionine-(S)-sulfoxide; Msr, methionine sulfoxide reductase C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
L-methionine-(S)-sulfoxide [Met-S-(O)] and L-methionine-(R)sulfoxide [Met-R-(O)]. Consequently, two types of Msr enzymes exist which reduce Met(O) in a stereospecific manner. Reduction of Met-S-(O) is catalyzed by MsrA, while reduction of the R-configured sulfoxide is mediated by MsrB [5, 6]. To study the stereospecificity of Met(O) reduction by Msr enzymes, the individual diastereomers 4-N,N-dimethylaminoazobenzene-4 -sulfonyl (dabsyl)-Met-S(O) and dabsyl-L-methionine-(R)-sulfoxide have been frequently used as substrates [7–9]. Furthermore, CE-based assays separating the diastereomers of dabsyl-Met(O) [10], 9-fluorenylmethyloxycarbonyl (Fmoc)-Met(O) [11] or the Nacetylated, C-terminal 2,4-dinitrophenyl (Dnp)-labeled peptide Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp [12] have been reported. CE has matured to one of the major separation technique for peptide analysis [13–15] including the separation of peptide enantiomers and diastereomers [16–19]. Furthermore, the successful separation of aryl and alkyl sulfoxide enantiomers [20, 21] as well as the sulfoxide enantiomers of the drugs albendazole [22] and thioridazine [23] has
† This paper is included in the virtual special issue sample preparatioin mass spectrometry available at the Journal of Separation Science website.
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been accomplished. Recently, the first separation of peptide diastereomers containing Met(O) has been described by our laboratory [12]. The diastereomers Ac-Lys-Ile-Phe-Met(O)Lys-Dnp were resolved by the use of a dual selector system composed of sulfated -CD and the achiral crown ether 15crown-5. Therefore, the aim of the present study was a further investigation of the dual selector system for Met(O) peptide diastereomer separation including several CDs as well as achiral crown ethers. Synthetic N-acetylated pentapeptides containing a Dnp label at the C terminus were studied.
2 Materials and methods 2.1 Chemicals The Met(O) peptide diastereomers, Ac-Lys-Asp-Met(O)Asp-Lys-Dnp, Ac-Lys-Asp-Met(O)-Asn-Lys-Dnp, Ac-LysAsn-Met(O)-Asp-Lys-Dnp, Ac-Lys-Val-Met(O)-Val-Lys-Dnp, Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp and Ac-Lys-Ile-Phe-Met(O)Lys-Dnp, were purchased from Peptide Specialty Laboratories (Heidelberg, Germany). -Cyclodextrin (-CD), ␥-CD, methyl--CD, 2-hydroxypropyl--CD, heptakis(2,6-diO-methyl)--CD, sulfopropyl--CD, sulfobutyl ether--CD, carboxymethyl-␥-CD and carboxymethyl--CD (CM--CD) were obtained from Cyclolab (Budapest, Hungary), while sulfated -CD, sulfated ␥-CD and Tris were from SigmaAldrich (Deisenhofen, Germany). Citric acid monohydrate, R 21 (7,13-diaza-1515-crown-5, 18-crown-6, Kryptofix crown-5, 1,4,10-trioxa-7,13-diazacyclopentadecane) and R 22 (4,13-diaza-18-crown-6, 1,7,10,16-tetraoxaKryptofix 4,13-diazacyclooctadecane) were purchased from MerckMillipore (Darmstadt, Germany). HPLC-grade acetonitrile and methanol were from VWR International (Darmstadt, Germany). All other chemicals were of the highest purity commercially available and used without further purification. Water was purified by a Milli-Q Direct 8 system (Merck-Millipore, Darmstadt, Germany).
the pH of 50 mM phosphoric acid with 1 M sodium hydroxide solution. All buffers were filtered (0.2 m) and sonicated for 5 min before use. 2 mM stock solutions of the peptides were prepared in water. Working solutions were obtained by a tenfold dilution with water before the injection. 2.3 Instrumentation All CE experiments were performed using a Beckman P/ACE MDQ CE system (Beckman Coulter, Krefeld, Germany) equipped with a UV-Vis diode array detector and a sample tray temperature control system set at 10⬚C. Instrument control and data evaluation were accomplished by the 32 KaratTM Software. A 40/50.2 cm, 50 m id, 363 m od fused-silica capillary (BGB Analytik Vertrieb, Schlossb¨ockelheim, Germany) was used. A new capillary was successively rinsed at a pressure of 138 kPa (20 psi) with 1 M sodium hydroxide for 20 min, water for 10 min, 0.1 M sodium hydroxide for 20 min, water for 10 min, and the BGE for 10 min. At the end of each day, the capillary was rinsed with 0.1 M sodium hydroxide for 10 min, water for 10 min and left in water overnight. Between analyses, the capillary was flushed with 0.1 M sodium hydroxide for 3 min, water for 2 min, and the BGE for 3 min. The separation voltage was 25 kV and the capillary temperature was set at 20⬚C. Hydrodynamic sample injection was performed at a pressure of 3.4 kPa (0.5 psi) for 4 s. UV detection was carried out at 214 nm either at the cathodic end or at the anodic end upon reversal of the separation voltage. 2.4 Calculations Peptide pI values were calculated using the GenScript Peptide Property Calculator (www.genscript.com/sslbin/site2/peptide_calculation.cgi). Peak resolution was calculated by the 32 KaratTM Software according to RS = 2(t2 – t1 )/(w1 +w2 ) where t1 and t2 are the migration times, and w1 and w2 are the widths of the peaks at the baseline.
2.2 BGEs and solutions
3 Results and discussion 50 mM Tris-citric acid buffers were prepared by dissolving the appropriate amount of Tris in water and adjusting the pH with 1 M citric acid before completing to the desired volume with water. For buffers containing a single selector, the appropriate amount of CD or crown ether was added to 25 mL of the respective Tris-citric acid buffer. After addition R diaza-crown ethers, the pH of the buffer of the Kryptofix was readjusted using a 1 M solution of citric acid. For the buffers containing two selectors, the appropriate amount of the crown ether was first added to 50 mL of the Tris-citric acid buffer. As in the case of single selector-containing buffers, the pH of the buffer was readjusted using a 1 M solution R diaza-crown of citric acid after the addition of Kryptofix ethers. Subsequently, the appropriate amount of a CD was added to 25 mL of the crown ether-containing Tris buffer. A 50 mM sodium phosphate buffer was prepared by adjusting C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.1 Screening of CDs for diastereomer separation Evaluation of the separation conditions was performed using the N-acetylated and C-terminal Dnp-labeled pentapeptides Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp, Ac-LysAsn-Met(O)-Asp-Lys-Dnp, Ac-Lys-Val-Met(O)-Val-Lys-Dnp, Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp and Ac-Lys-Ile-Phe-Met(O)Lys-Dnp. Assuming that the oxidation of Met does not affect the pI of peptides, Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp can be considered a neutral peptide with a pI of about 7.0. A pI value of 8.9 was calculated for Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp, while the values of the remaining peptides were 10.5 (Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp) and 10.8 (Ac-Lys-ValMet(O)-Val-Lys-Dnp and Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp). Thus, all peptides are positively charged in the acidic pH www.jss-journal.com
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range and, except for Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp, also in the slight alkaline pH range. Therefore, the separation was investigated in a fused-silica capillary in the pH range 2.5–9.5 using 50 mM Tris-citric acid buffers or phosphate-based buffers. However, no separation of the peptide diastereomers was observed under these conditions. Subsequently, neutral and charged CDs including native -CD and ␥-CD, methyl--CD, 2-hydroxypropyl-CD, heptakis(2,6-di-O-methyl)--CD, sulfopropyl--CD, sulfobutyl ether--CD, sulfated -CD, sulfated ␥-CD, carboxymethyl--CD (CM--CD) and carboxymethyl-␥-CD were evaluated as buffer additives at a concentration of 15 mg/mL in a 50 mM Tris-citric acid buffer at pH 2.5 and pH 8.0. At pH 2.5, only a very limited partial separation was observed for Ac-Lys-Ile-Phe-Met(O)-LysDnp and Ac-Lys-Val-Met(O)-Val-Lys-Dnp in the presence of CM--CD. None of the analytes could be detected within 100 min under normal polarity of the separation voltage in the presence of sulfated -CD even when reducing the concentration of the selector to 2 mg/mL. Consequently, the concentration of sulfated -CD was raised to 30 mg/mL and the polarity of the separation voltage was reversed. Under these conditions, Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp and Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp could be detected within 10 min but no peptide diastereomer separation was observed. At pH 8.0, partial separation of the diastereomers of Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp, Ac-Lys-Glu-Met(O)-LysLys-Dnp, Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp and Ac-Lys-AsnMet(O)-Asp-Lys-Dnp was noted in the presence of 15 mg/mL sulfated -CD with normal polarity of the separation voltage. Baseline separation (Rs > 1.5) of Ac-Lys-Ile-Phe-Met(O)-LysDnp and Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp was achieved in a BGE containing 20 mg/mL sulfated -CD, 50 mM Tris-citric acid buffer, pH 7.5. The other peptide diastereomers could not be baseline resolved no matter what sulfated -CD concentration or buffer pH was investigated. All other investigated CDs proved to be ineffective as BGE additives for achieving at least a partial separation under the experimental screening conditions.
3.2 Screening of the dual system of achiral crown ethers and CDs It has been reported in the literature that the addition of achiral crown ethers can enhance enantioseparations of primary amines including amino acids. 18-crown-6 [24–26] or diaza-crown ethers [27, 28] have been used in combination with native -CD or neutral -CD derivatives. These enantioseparations are assumed to be based on the formation of a sandwich-type complex between the analyte enantiomers and the respective CDs and crown ethers [29, 30]. Consequently, the crown ethers 18-crown-6 and 15-crown-5 as well R 22 (4,13-diaza-18-crownas the diaza-crown ethers Kryptofix R 6) and Krpytofix 21 (7,13-diaza-15-crown-5) were studied in combination with -CD, CM--CD and sulfated -CD using Tris-citric acid buffers with a pH of 2.5 and 8.0 as BGEs. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The concentration of the CDs was set at 10 mg/mL, while 18-crown-6 and 15-crown-5 were tested at a concentration of R 15 mM and the nitrogen-containing Krpytofix crown ethers at 8 mM. The results in comparison with the sole use of CDs as additives are summarized in Supporting Information Tables S1 (pH 2.5) and S2 (pH 8.0). It should be noted that addition of a crown ether to the BGE in the absence of a CD in concentrations up to 20 mM did not result in any separation. The neutral crown ethers, 18-crown-6 and 15-crown-5, displayed a synergistic effect on the diastereomer separation in combination with CM--CD at pH 2.5 and with sulfated -CD at pH 8.0. While partial separations were observed at pH 2.5, baseline separations could be observed for AcLys-Ile-Phe-Met(O)-Lys-Dnp and Ac-Lys-Glu-Met(O)-Lys-LysDnp with sulfated -CD and the crown ethers. Migration times increased in the presence of the crown ethers compared to the sole presence of CDs indicating that a ternary complex may be formed between peptide diastereomers, CDs and crown ethers as derived for amine analytes [24, 29, 30]. No separation could be observed in combination with native -CD even at concentrations of up to 25 mg/mL of the CD. The pKa values of diaza-crown ethers are about 9 [31], so that the compounds are protonated at the pH values studied. Interestingly, none of the analytes could be detected at R 22 under both, norpH 2.5 in the presence of Kryptofix mal and reversed polarity of the separation voltage. A similar behavior has also been observed by Iv´anyi and colleagues when attempting the enantioseparation of Trp by the comR 22 and neutral CD derivatives [28]. bination of Kryptofix In contrast, partial diastereomer separation of several pepR 21. It may be speculated tides were observed for Kryptofix R that the protonated Kryptofix 22 is adsorbed to the capillary wall, thus, modifying the EOF, while this occurs to a R 21 crown ether. lesser extent in case of the smaller Kryptofix Diaza-crown ethers have been used for EOF modification and reversal for the separation of inorganic ions as bifunctional modifiers [32, 33]. Migration times increased as compared to the presence of the neutral crown ethers despite the fact that the charge of the complexes should be higher in the case of the protonated diaza-crown ethers. This further indicates that the diaza-crown ethers induced an EOF directed toward the anode. Finally, the diastereomers of Ac-Lys-Ile-Phe-Met(O)R 21 Lys-Dnp were resolved by the combination of Kraptofix and -CD at pH 2.5. A significant synergistic effect between the CDs and the diaza-crown ethers was noted at pH 8.0. Except for Ac-LysAsp-Met(O)-Asp-Lys-Dnp with the approximate pI of 7.0, all other peptide diastereomers were at least partially separated using CM--CD. Effective separations were found in the presence of sulfated -CD with baseline resolution RS > 5 for Ac-Lys-Ile-Phe-Met(O)-Lys-Dnp in the presence of both diaza-crown ethers. The diastereomers of this peptide were even resolved when -CD was used as CD component. Figure 1 illustrates the effect of the crown ethers on the diastereomer separation of Ac-Lys-Glu-Met(O)-LysLys-Dnp under the screening conditions at pH 8.0 as an example. www.jss-journal.com
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J. Sep. Sci. 2014, 00, 1–7 Figure 1. Effect of CDs and crown ethers as BGE additives on the separation of the diastereomers of Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp. Experimental conditions: 40/50.2 cm, 50 m id fused-silica capillary, 50 mM Tris-citric acid buffer, pH 8.0, 25 kV, 20⬚C, detection at 214 nm. Buffer additives (A) 10 mg/mL sulfated -CD, (B) 10 mg/mL sulfated -CD and 15 mM 15-crown-5, (C) 10 mg/mL sulfated -CD and 15 mM 18-crown-6, (D) 10 mg/mL R sulfated -CD and 8 mM Kryptofix 21, and (E) R 10 mg/mL sulfated -CD and 8 mM Kryptofix 22.
3.3 Effect of CD and crown ether concentration in the dual system The effect of different CD and crown ether concentrations on the separation of the peptide diastereomers was studied R 22 as well as sulfated using 18-crown-6 and Kryptofix CD, except for Ac-Lys-Val-Met(O)-Val-Lys-Dnp. In this case, CM--CD was applied because it proved to be an effective additive for the diastereomer separation of this peptide in the screening experiments. The experiments were conducted at pH 8.0 because efficient separations were observed at this pH in contrast to pH 2.5. The concentration of one type of selector was fixed while the concentration of the second selector was varied. The results are summarized in Fig. 2. Generally, with a few exceptions, an increase of peak resolution and migration time can be observed upon increasing the concentration of a selector while keeping the other selector constant. In the case of Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp an optimum appears to exist for a CD concentration (with constant crown ether concentrations) of about 15 mg/mL because peak resolution decreased when further increasing the CD concentration (Fig. 2B and D). Only partial resolution of the diastereomers of Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp was R 22 < 10 mM, while observed at concentrations of Kryptofix no separation was observed at higher concentrations. In contrast to the other systems, migration analyte times R 22-based BGE upon addecreased in case of the Kryptofix dition of 5 mg/mL of the CDs and subsequently increased at higher CD concentrations (Fig. 2H). This decrease did not correlate with a general decrease in peak resolution (Fig. 2D). As stated above, it may be speculated that the positively charged diaza-crown ether is adsorbed to the capillary wall resulting in a slow EOF directed toward the anode. This can be counteracted by the presence of negatively charged CDs such as sulfated -CD or CM--CD resulting in a faster migration of the positively charged analytes to the detector at the cathodic end of the capillary. The subsequent increase of the migration times at higher CD concentrations can be explained by the increased analyte complexation and increased viscosity of the buffer at higher CD concentrations.
3.4 Effect of buffer pH The effect of the pH of the Tris-citric acid-based electrolyte was studied in the pH range 2.5 to 9.5 in BGEs C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
containing 15 mg/mL CM--CD and 20 mM 18-crown-6 or R 22. The diaza-crown ether is only partially 10 mM Kryptofix protonated above pH 9.0, while CM--CD will be protonated at pH < 3–4. Ac-Lys-Val-Met(O)-Val-Lys-Dnp was used as model peptide because separations in CM--CD-based BGEs have been observed during the screening experiments. The peptide is positively charged over the entire pH range studied (pI of approximately 10.8). Increasing the pH of the BGE containing CM--CD and 18-crown-6 resulted in a deterioration of the diastereomer separation with a concomitant decrease of migration times (data not shown). No separation could be observed at pH values above 4. This may be explained by the increased EOF at higher pH values leading to short migration times, which did not allow sufficient time for analyte-selector interactions. R 22, long For the combination CM--CD and Kryptofix migration times were observed at pH values < 6.5. The dependence of the resolution of the diastereomers of Ac-LysVal-Met(O)-Val-Lys-Dnp in the pH range 6.5–9.5 is depicted in Fig. 3. The highest resolution was found at pH 7.5 to 8. Further increase of the pH resulted in a loss of resolution. Migration times also significantly decreased, e.g. from about 45 min at pH 7.5 to about 25 min at pH 8.5 with a concomitant decrease of resolution from 2.4 to 2.1. Thus, the recommended pH for the separation would be pH 8.0 to 8.5 as a compromise of resolution and analysis time. It may be speculated that the decreasing migration times (and loss of R 22 is less proresolution) are due to the fact that Kryptofix tonated at pH 8–8.5 so that interaction of crown ether with the capillary wall is weakened resulting in a change of the EOF directed toward the cathode. The effect of the change of the charge of the selectors as a function of buffer pH on the formation of the ternary complex with the positively charged peptide cannot be concluded from the present data.
3.5 Diastereomer separation of a peptide mixture The simultaneous separation of the diastereomers of Ac-LysAsp-Met(O)-Asp-Lys-Dnp, Ac-Lys-Asp-Met(O)-Asn-Lys-Dnp and Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp was also studied. The peptides differ in an exchange of the acidic amino acid Asp with the neutral amino acid Asn either N- or C-terminal to Met(O), which results in a basic pI value of about 8.9 versus the pI of about 7.0 of the peptide containing two Asp residues. The electropherograms are shown in Fig. 4. In the www.jss-journal.com
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Figure 2. Effect of the concentration of CDs and R the crown ethers, 18-crown-6 and Kryptofix 22, on (A–D) resolution of Met(O) peptide diastereomers and (E–H) migration time of the first migrating diastereomer. Experimental conditions: 40/50.2 cm, 50 m id fusedsilica capillary, 50 mM Tris-citric acid buffer, pH 8.0, 25 kV, 20⬚C, detection at 214 nm. CM-CD was used for the separation of Ac-Lys-ValMet(O)-Val-Lys-Dnp while sulfated -CD was used for the separation of the other peptides. The concentration of the CD was fixed at 10 mg/mL when varying the crown ether concentrations (A, C, E, G). The concentrations of 18-crown-6 was fixed at 15 mM (B, R F) and Kryptofix 22 at 8 mM (D, H) when varying the CD concentration. 䊉, Ac-Lys-AspMet(O)-Asp-Lys-Dnp; 䉱, Ac-Lys-Val-Met(O)-ValLys-Dnp; 䊊, Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp; , Ac-Lys-Glu-Met(O)-Lys-Lys-Dnp; , Ac-LysIle-Phe-Met(O)-Lys-Dnp.
Figure 3. pH-Dependence of the separation of the Ac-LysVal-Met(O)-Val-Lys-Dnp diastereomers. Experimental conditions: 40/50.2 cm, 50 m id fused-silica capillary, 50 mM Tris-citric acid R buffer, 15 mg/mL CM--CD, 10 mM Kryptofix 22, 25 kV, 20⬚C, detection at 214 nm.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
absence of CDs and/or crown ethers, the three peptides migrated as a single peak. The peptides were separated in the presence of 12 mg/mL sulfated -CD but only partial diastereomer separation of the Asn-containing peptides were observed (Fig. 4A). As expected, improvement of the diastereomer resolution could be achieved by further addition of the R 22 (Fig. 4C). crown ethers, 18-crown-6 (Fig. 4B) or Kryptofix However, the separation of the diastereomers of Ac-Lys-AspMet(O)-Asp-Lys-Dnp was still incomplete. Thus, addition of organic solvents was considered which may affect the interactions between analytes and BGE additives. Subsequently, 15% v/v of acetonitrile or methanol were added to the BGE. While the presence of methanol did not affect the diastereomer separation and significantly prolonged migration times,
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been combined with the crown ethers. The present data on peptide diastereomer separations indicate that crown ethers can also be effectively combined with negatively charged CDs for stereoisomer separations. The financial support of Q. Zhu by the China Scholarship Council (CSC) is gratefully acknowledged. The authors have declared no conflict of interest.
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Figure 4. Electropherograms of separation of the peptide diastereomers Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp, Ac-Lys-AspMet(O)-Asn-Lys-Dnp, and Ac-Lys-Asn-Met(O)-Asp-Lys-Dnp. Experimental conditions: 40/50.2 cm, 50 m id fused-silica capillary, 50 mM Tris buffer, pH 8.0, 25 kV, 20⬚C, detection at 214 nm. Buffer additives are (A) 12 mg/mL sulfated -CD, (B) 12 mg/mL sulfated -CD plus 25 mM 18C6, (C) 12 mg/mL sulfated -CD plus 6 mM R Kryptofix 22, (D) 12 mg/mL sulfated -CD, 25 mM 18-crown-6, plus 15% acetonitrile.
addition of acetonitrile significantly improved the resolution of Ac-Lys-Asp-Met(O)-Asp-Lys-Dnp as shown in Fig. 4D for the combination of sulfated -CD and 18-crown-6.
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4 Concluding remarks
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A dual selector system has been developed for the separation of the diastereomers of Met(O)-containing peptides by CE. Combinations of the crown ethers 18-crown-6 and 15-crown-5 R R 21, Kryptofix 22 as well as the diaza-crown ethers Kryptofix and -CD derivatives were evaluated. Especially dual systems containing sulfated -CD or CM--CD and a diaza-crown ether could be used for the resolution of the Met(O) peptide diastereomers in pH 8.0 BGEs. This is interesting to note because both, peptide and diaza-crown ether, are at least partially positively charged at this pH. The present data further support the additive effect of CDs and crown ethers for aminecontaining analytes as reported for enantiomer separations in the literature [24–30]. In these studies, neutral CDs have C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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