Capillary Zone Electrophoresis for the Analysis of Peptides: Fostering

Laboratory Experiment pubs.acs.org/jchemeduc

Capillary Zone Electrophoresis for the Analysis of Peptides: Fostering Students’ Problem-Solving and Discovery Learning in an Undergraduate Laboratory Experiment Jessica C. Albright and Douglas J. Beussman* Department of Chemistry, St. Olaf College 1520 St. Olaf Avenue, Northfield, Minnesota 55057, United States S Supporting Information *

ABSTRACT: Capillary electrophoresis is an important analytical separation method used to study a wide variety of samples, including those of biological origin. Capillary electrophoresis may be covered in the classroom, especially in advanced analytical courses, and while many students are exposed to gel electrophoresis in biology or biochemistry laboratories, capillary electrophoresis is not as commonly found in undergraduate lab curricula. Published lab experiments that incorporate capillary electrophoresis include the analysis of caffeine in beverages and cations in water samples but not of more biological samples such as a peptide mixture. This paper introduces a peptide separation based on capillary zone electrophoresis that is both qualitative and quantitative. It describes the separation of four small peptides where two of the analytes have similar migration times, forcing students to think about how they can best identify which peak corresponds to which analyte, while exposing them to a bioanalytical application of capillary electrophoresis. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Bioanalytical Chemistry, Electrophoresis, Instrumental Methods, Proteins/Peptides

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INTRODUCTION Capillary electrophoresis (CE) is an important analytical and bioanalytical separation method. Although commercially it is a much younger field than other separation techniques, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC), it has become the standard method used for DNA sequencing in both biological and forensic laboratories.1−3 It is also routinely used for the analysis of small anions and cations.4 Indeed, CE is routinely used in research applications for the analysis of peptides.5−8 Microfluidic CE devices are often connected to electrospray mass spectrometers for proteomic analysis.9−11 Capillary electrophoresis is covered in most analytical textbooks, and several laboratory experiments have been developed to demonstrate the power of this technique.12 These include laboratories aimed at determining the amount of caffeine in various foods and beverages,13−15 separation of barbiturates,16 analysis of cations in soil and water samples,17−20 studying the reaction products from an organic chemistry experiment,21,22 the analysis of various foods and food dyes,23 and analgesic formulations.24 Laboratories with biological applications include determining protein amino acid composition,25 determining the amino acid composition of a dipeptide using the related micellar electrokinetic capillary chromatography technique,26 carbohydrate analysis,27 and determining the diffusion constant of dopamine.28 The experiment we present here focuses on the separation and quantitation of several small peptides using capillary zone © XXXX American Chemical Society and Division of Chemical Education, Inc.

electrophoresis (CZE), an area not previously described in the undergraduate curriculum to our knowledge. CZE is the simplest form of CE, with the migration rates of analytes based on their electrophoretic mobilities and the overall capillary electro-osmotic flow. While peptides are routinely separated by HPLC,29 the ability to separate peptides using CE is also important for students to understand. It provides them with a hands-on experience to a second separation method that they can compare and contrast to more established HPLC methods that they have learned about in previous courses. While students in this class do not do both an HPLC and a CE separation, most of them have used HPLC in a previous lab so that they can consider differences in sample amount, buffer/ mobile phase amounts, ease of use, analysis time, and other factors between the two separation techniques. It is also important for students to appreciate the significant reduction in solvent/mobile phase that is inherent in CE as compared to HPLC. While CE does require very large voltages for effective analyte separation, the small amount of buffer needed makes it a significantly “greener” separation technique compared to most HPLC separation methods. HPLC often uses organic solvents as part of the mobile phase, with flow rates up to 1 mL per minute over the course of the entire Received: June 24, 2016 Revised: February 1, 2017

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DOI: 10.1021/acs.jchemed.6b00463 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. Separation of bradykinin (4.937 min), angiotensin 1 (5.220 min), leucine enkephalin (8.297 min), and methionine enkephalin (8.803 min).

separation. Often a mobile phase reservoir of several hundred milliliters is filled for an afternoon lab. In contrast, capillary electrophoresis requires much less than a milliliter of buffer for a single analysis. The buffer reservoirs may contain 1 to 10 mL of buffer, sufficient for an entire afternoon lab session.

were also given an unknown sample and told that it contained 1−4 of the standards and that they needed to determine which peptide(s) were in the unknown and the concentration of each. Students were told that the linear working range for the experiment was between 0.01 and 1.0 nmol/μL for each peptide and that dilutions should be made with 18 MΩ·cm water. They were instructed to load 10−25 μL of sample into the bottom of each plastic conical CE vial, making sure that there were no air bubbles in the bottom of the vial. The instrument data analysis software is used to determine peak heights for the quantitation portion of the experiment. Since migration rates affect peak areas, using areas directly can cause issues with quantitation. An alternative measurement uses the ratio of area to migration time to account for the widening of peaks as the migration time increases. Students use Microsoft Excel to obtain a linear fit between peptide concentration and peak heights (or area/time ratios) and use the slope and intercept to determine the concentration of their unknown peptide(s).

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EXPERIMENTAL PROCEDURES All chemicals and peptide samples were purchased from SigmaAldrich. Conditioning and buffer solutions were made using 18 MΩ·cm water. Peptides were stored in the freezer (−20 °C) following given guidelines. For each peptide, a 1 nmol/μL (1 mM) lab solution (1 mL total) was prepared from stock using nanopure (18 MΩ·cm) water. Peptide “unknown” samples and all student peptide calibration samples were made by further dilution of the lab solutions with 18 MΩ·cm water. An uncoated capillary with 50 μm inner diameter and an overall length of 64.5 cm (detector window after 56 cm) was used. This column was installed in an Agilent capillary electrophoresis instrument, although any CE instrument should be able to provide comparable results. Prior to lab, the column was conditioned by flushing for 10 min each with acetonitrile, 0.1 M hydrochloric acid, 1.0 M sodium hydroxide, and 18 MΩ· cm water. A final flush using 50 mM acetic acid (pH 3.0 ± 0.1 as measured with a pH electrode) was performed for 20 min prior to use. When possible, the instrument was conditioned the night before the experiment, and the final 50 mM acetic acid solution was allowed to remain in the capillary overnight. Students set up their sample analyses and ran each sample. Each analysis consisted of a 5 min preconditioning flush with 50 mM acetic acid solution flushing into an outlet vial. The background electrolyte was the same 50 mM acetic acid solution. Analyses were done at a constant temperature of 25 °C, with a run voltage of 30 kV and a run time of 20 min. Samples were injected under constant pressure of 50 mbar for 6 s. Peptide absorbance signals were measured at 192 nm. A more detailed description of the instrumental parameters can be found in the Supporting Information. Students were given four known standard solutions of angiotensin 1, bradykinin, leucine enkephalin, and methionine enkephalin, each at 1 nmol/μL (1 mM) concentration. They

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HAZARDS

Gloves should be worn when handling samples and solvents to prevent skin contact. Goggles should also be worn when working with all solutions. Commercial capillary electrophoresis instruments have built-in safety interlocks, so there is relatively low risk associated with the operation of the instrument. Care should be taken to avoid contact with all solutions. The HCl and NaOH, used in the capillary conditioning, are a strong acid and base, respectively, and care must be taken when handling these solutions. Acetonitrile, also used in the capillary conditioning, is flammable and toxic. The run electrolyte (50 mM acetic acid pH 3.0 ± 0.1) is flammable and corrosive. Even though only a few milliliters of solution is used, all safety protocols (goggles and gloves) should be followed. The peptides used are classified as nonhazardous and have no GHS label elements. Students are instructed, however, to wear safety goggles and gloves when handling the samples as part of safe laboratory practices. B




Figure 2. Leucine enkephalin standard added to a sample containing methionine enkephalin.

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RESULTS AND DISCUSSION This experiment has been part of the Bioanalytical Chemistry course for six course offerings over the past 12 years, with an average of 11 students enrolled each time the course is taught. Students were given an unknown sample that contained one of four possible peptides. If each of the four standards were analyzed separately, each electropherogram would have contained a single major peak. Migration times could be used to correlate unknown samples with the peptide standards. Figure 1 shows the separation of all four peptides. No instruction was given as to how to determine the peptide(s) in each unknown. Ideally, each standard would have been run individually, followed by the unknown and migration times used to determine the peptide(s) in the unknown, or at least to narrow it down. Once the peptide had been qualitatively identified, a working curve could be made using the standard solution for each peptide in the sample and the concentration of each peptide determined in the unknown using peak height data. By identifying the peptide(s) in the sample first, the need to make and analyze all of the calibration standards for peptides not in the unknown could be avoided. In practice, each unknown had one peptide, meaning that at least five samples needed to be run to determine the identity (four standards and the unknown) and four to five calibration standards for quantitative analysis, for a total of 9−10 samples. Since each sample took a little over 30 min for the preconditioning, sample loading, and separation, each student required about 5 h of instrument time. Since the instrument had an autosampler tray, students routinely made their samples, ran the qualitative samples, and then made the calibration standards and let the quantitation samples run overnight. While all four peptides are usually not run at the same time, as it would be impossible to determine which migration time corresponded to which peptide, Figure 1 demonstrates that all four peptides are able to be separated. While the electroosmotic flow was not directly measured in this experiment, it would be expected to be fairly low since the run electrolyte pH is 3.0 ± 0.1. This will limit the charges on the capillary wall, decreasing the double layer and the electro-osmotic flow rate. Therefore, the separation was mainly driven by differences in electrophoretic flow rates between the analytes. The low pH of the run electrolyte also helps decrease peptide adsorption to the

fused silica capillary walls. This adsorption can be caused by hydrogen bonding effects as well as electrostatic attraction and can lead to peak broadening.30 A low electrolyte pH minimizes the charge on the capillary wall, decreasing but not eliminating the attraction of biological molecules to the capillary walls. As can be seen in Figure 1, the migration times of angiotensin 1 and bradykinin are quite similar, as are the migration times for Leu-enkephalin and Met-enkephalin. While each of these pairs can be separated, slight migration time shifts between runs can cause confusion as to which of the peptides are in the unknown. For example, if the angiotensin 1 peak were to shift 17 s earlier, it would overlap with the bradykinin peak. If it were to shift less than 17 s, the peaks would still be partially resolved, but it might be difficult to assign them based solely on migration time differences of a few seconds. These migration shifts can be caused by a variety of factors, including slight instrument timing shifts, slight differences in applied voltage or column temperature, ionic strength differences between samples (ionic strength affects the electro-osmotic flow and thus the migration time), or differences in run electrolyte composition between runs. Sample adsorption to the capillary walls can also affect the electro-osmotic flow rate, leading to changes in migration times. As noted above, this can be minimized by using low pH electrolyte solutions, but any slight pH changes between standards and the peptide unknown can result in the observed migration shifts. While migration shifts large enough to prevent peptide identification were not seen for all analyses, large enough shifts between standards and unknowns were seen in approximately half the samples such that assigning the unknown peptide identification based solely on migration time could not be done. It was often confusing as to how to assign the unknown if these time shifts were observed. It should be noted that this confusion can be avoided by using only two peptides instead of all four and choosing two that have a large difference in migration times. When all four peptides were used, no instruction was initially given as to how to determine which of the two peptides with close migration times corresponds to the unknown. After some thought, it was eventually realized that if one of the standards was mixed with the unknown, one peak would be observed if the same compound was added as was in the unknown sample or two peaks if the other peptide C




was added to the unknown. Sometimes gentle guidance was needed to reach this realization by asking why two different peptides had different migration rates, what factors could cause the time shifts, and if these factors were compound specific or if they affected all compounds in a given injection. Once it was realized that adding one standard to the unknown would yield either a single peak or two peaks, the unknown could quickly be identified with one additional CE injection. Figure 2 shows student results after the addition of leucine enkephalin standard to a methionine enkephalin unknown sample in a 2:1 ratio, respectively, resulting in two peaks. After the migration order of the four peptides had been determined, students were asked if they could provide a rational explanation for what they observed. Some students thought about the size of the peptides, while others thought about charge. Eventually, most realized that there is not much size difference between these peptides, especially the enkephalins, and they settled on looking at the charge via predicted isoelectric points. Since students had used the program for other portions of the course, they generally used the commercial software program GPMAW to calculate isoelectric points based on the amino acid sequence,31 although there are a variety of Web-based predictors of isoelectric point available.32,33 Once the isoelectric points were determined, the increasing migration time could easily be related to decreasing isoelectric point, as shown in Table 1. Since the run electrolyte has a pH of 3.0 ± 0.1, all four analytes will be positively charged, leading to electrophoretic flow down the capillary.

Table 2. Student Results from the Eight Students Enrolled in the 2015 Course Offering

Table 1. Observed Migration Order versus Predicted Isoelectric Point

Figure 3. Calibration curves for four peptides, showing R2 values for five measurements between 0.01 and 1 nmol/μL.

peptide

migration order

isoelectric point

bradykinin angiotensin 1 Leu-enkephalin Met-enkephalin

first second third fourth

12.5 7.96 5.68 5.58

reported unknown

actual unknown

Leu-enkephalin angiotensin 1 Met-enkephalin Met-enkephalin angiotensin 1 Leu-enkephalin Met-enkephalin Met-enkephalin

Leu-enkephalin angiotensin 1 Met-enkephalin Met-enkephalin angiotensin 1 Leu-enkephalin Met-enkephalin Met-enkephalin

reported concentration (nmol/μL) (mM)

actual concentration (nmol/μL) (mM)

± ± ± ± ± ± ± ±

0.15 0.40 0.25 0.25 0.40 0.15 0.25 0.25

0.16 0.38 0.28 0.23 0.43 0.17 0.28 0.21

0.02 0.04 0.03 0.02 0.04 0.02 0.03 0.02

peptides to which they had narrowed it down. Students were usually allowed to think about how best to accomplish this for an hour or two before the instructor started giving hints such as “what would happen if you had two different peptides mixed together?” or “what would you observe if you mixed two aliquots of the same peptide together?” Once a few students in the lab had completed this experiment, other students quickly learned from them how to best determine their unknown. Historically, nearly 95% of all unknown peptides have been correctly identified, and over 85% of the concentrations have been calculated within a 10% error of what they were made. Procedures and results were summarized in a written laboratory report at the end of the lab. Since this experiment was part of a month-long Bioanalytical course in which students were only taking this one course and thus could be in lab for several hours each day, this experiment was completed individually to maximize hands-on access to the instrument. For more conventional laboratories that meet once a week, small groups could be used to complete this experiment. Alternatively, fewer possible peptide unknowns could be used, reducing the number of samples needed to be run in order to identify the unknown peptide. Another method for reducing the amount of time need to complete the experiment would be to allow the entire lab section to use the same standards so that only one set of standard migration times is needed and/or to use a single calibration curve for the entire lab section so that not every student would need to make and run five standard concentrations. Pedagogically, the goals of this experiment were to allow students to learn more about capillary electrophoresis, an

Once the unknowns had been identified, a standard working curve was prepared by diluting the appropriate peptide standard. How many calibration points to use was not dictated by the instructor. Generally, four or five were suggested, but if less than four or more than six were proposed, the instructor usually suggested a more reasonable number. Lectures previously indicated that, for capillary electrophoresis, peak height or the ratio of peak area to migration time is often a better measure of concentration than is peak area due to sample plugs moving at different rates past the detector window. A slower moving sample plug will have the same height as a faster sample plug of the same concentration, but the slower sample will be wider since it moves past the detector more slowly and will therefore have a larger area than the faster sample. In practice, these differences are often negligible, but encouragement was still given to use either peak height or the area/time ratio to construct the calibration curves and determine the concentration of the unknown sample. In practice, virtually identical results were obtained using peak heights as using area/ time ratios. Table 2 shows representative student data, with the calibration curves for each shown in Figure 3. Ultimately, all unknowns were correctly identified using the strategy of adding a standard to the unknown described above. Initially, most students struggled to come up with this strategy, asking the instructor how to determine their unknown from the two D




(4) Koenka, I. J.; Mai, T. D.; Hauser, P. C.; Saiz, J. Simultaneous separation of cations and anions in capillary electrophoresis − recent applications. Anal. Methods 2016, 8 (7), 1452−1456. (5) Tetala, K. K. R.; Vijayalakshmi, M. A. A review on recent developments for biomolecule separation at analytical scale using microfluidic devices. Anal. Chim. Acta 2016, 906, 7−21. (6) Kasicka, V. Recent developments in capillary and microchip electroseparations of peptides (2013 − middle 2015). Electrophoresis 2016, 37 (1), 162−188. (7) Ali, I.; Al-Othman, Z. A.; Al-Warthan, A.; Asnin, L.; Chudinov, A. Advances in chiral separations of small peptides by capillary electrophoresis and chromatography. J. Sep. Sci. 2014, 37 (18), 2447−2466. (8) Nuchtavorn, N.; Suntornsuk, W.; Lunte, S. M.; Suntornsuk, L. Recent applications of microchip electrophoresis to biomedical analysis. J. Pharm. Biomed. Anal. 2015, 113, 72−96. (9) Heemskerk, A. M.; Deelder, A. M.; Mayboroda, O. A. CE-ESI-MS for bottom-up proteomics: Advances in separation, interfacing and applications. Mass Spectrom. Rev. 2016, 35 (2), 259−271. (10) Pejchinovski, M.; Hrnjez, D.; Ramirez-Torres, A.; Bitsika, V.; Mermelekas, G.; Vlahou, A.; Zuerbig, P.; Mischak, H.; Metzger, J.; Koeck, T. Capillary zone electrophoresis on-line coupled to mass spectrometry: A perspective application for clinical proteomics. Proteomics: Clin. Appl. 2015, 9 (5−6), 453−468. (11) Ruige, W.; Fung, Y. S. Microfluidic chip-capillary electrophoresis device for the determination of urinary metabolites and proteins. Bioanalysis 2015, 7 (7), 907−922. (12) Holland, L. A. Capillary electrophoresis: Focus on undergraduate laboratory experiments. J. Chem. Educ. 2011, 88 (3), 254− 256. (13) Beckers, J. L. The determination of caffeine in coffee: Sense or nonsense? J. Chem. Educ. 2004, 81 (1), 90−93. (14) McDevitt, V. L.; Rodriguez, A.; Williams, K. R. Analysis of soft drinks: UV spectrophotometry, liquid chromatography, and capillary electrophoresis. J. Chem. Educ. 1998, 75 (5), 625−629. (15) Conte, E. D.; Barry, E. F.; Rubinstein, H. Determination of caffeine in beverages by capillary zone electrophoresis - An experiment for the undergraduate analytical laboratory. J. Chem. Educ. 1996, 73 (12), 1169−1170. (16) Contradi, S.; Vogt, C.; Rohde, E. Separation of enantiomeric barbiturates by capillary electrophoresis using a cyclodextrincontaining run buffer. J. Chem. Educ. 1997, 74 (9), 1122−1125. (17) Pursell, C. J.; Chandler, B.; Bushey, M. M. Capillary electrophoresis analysis of cations in water samples - An experiment for the introductory laboratory. J. Chem. Educ. 2004, 81 (12), 1783− 1786. (18) Gruenhagen, J. A.; Delaware, D.; Ma, Y. F. Quantitative analysis of non-UV-absorbing cations in soil samples by high-performance capillary electrophoresis - An experiment for undergraduate instrumental analysis laboratory. J. Chem. Educ. 2000, 77 (12), 1613−1616. (19) Hage, D. S.; Chattopadhyay, A.; Wolfe, C. A. C.; Grundman, J.; Kelter, P. B. Determination of nitrate and nitrite in water by capillary electrophoresis - An undergraduate laboratory experiment. J. Chem. Educ. 1998, 75 (12), 1588−1590. (20) Janusa, M. A.; Andermann, L. J.; Kliebert, N. M.; Nannie, M. H. Determination of chloride concentration using capillary zone electrophoresis - An instrumental analysis chemistry laboratory experiment. J. Chem. Educ. 1998, 75 (11), 1463−1465. (21) Mills, N. S.; Spence, J. D.; Bushey, M. M. Capillary electrophoresis analysis of substituted benzoic acids - An experiment for the organic synthesis laboratory. J. Chem. Educ. 2005, 82 (8), 1226−1228. (22) Welder, F.; Colyer, C. L. Using capillary electrophoresis to determine the purity of acetylsalicylic acid synthesized in the undergraduate laboratory. J. Chem. Educ. 2001, 78 (11), 1525−1527. (23) Yuen, P. K.; Goral, V. N. Low-Cost Rapid Prototyping of Whole-Glass Microfluidic Devices. J. Chem. Educ. 2012, 89 (10), 1288−1292.

important but often not covered separation technique. By providing a hands-on CE experience, students were allowed to gain a deeper understanding of how the technique works. The described experiment also had students analyze an unknown sample both qualitatively and quantitatively and had them preparing a calibration curve. Finally, by including peptides with very similar migration times, students were forced to think about a strategy for determining which of the two peptides they had in their unknown. Since the vast majority of students correctly identified their unknown, with most calculating a concentration quite close to the actual concentration, it can be argued that the qualitative and quantitative goals of this experiment were met. As most students had not heard of capillary electrophoresis before this experience and most were able to correctly describe the technique on a final exam, the broader goal of understanding CE was also satisfied.

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CONCLUSIONS This experiment provides an introduction to the use of capillary electrophoresis for both qualitative and quantitative applications. While capillary electrophoresis may not be used as often as other separation methods, it is still an important technique to which to expose students. This experiment presents an alternative to HPLC for the separation and identification of small peptide samples and could therefore be of interest not only to Analytical or Instrumental Analysis laboratories but also to the Biochemistry lab curriculum. Depending on which peptides students are given, the instructor can also show students how two compounds with similar but distinct migration (or elution) times can be distinguished from each other. Finally, this experiment represents a “greener” alternative to HPLC separation, which often uses significant amounts of solvents for the mobile phase.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00463. Copy of the student handout; instructor notes that may be helpful for others interested in adopting a similar experiment in their curriculum (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Douglas J. Beussman: 0000-0002-5366-6960 Notes

The authors declare no competing financial interest.

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REFERENCES

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(24) Thompson, L.; Veening, H.; Strein, T. G. Capillary electrophoresis in the undergraduate instrumental analysis laboratory: Determination of common analgesic formulations. J. Chem. Educ. 1997, 74 (9), 1117−1121. (25) Weber, P. L.; Buck, D. R. Capillary electrophoresis − A fast and simple method for the determination of the amino-acid-composition of proteins. J. Chem. Educ. 1994, 71 (7), 609−612. (26) Strein, T. G.; Poechmann, J. L.; Prudenti, M. Micellar electrokinetic capillary chromatography in the undergraduate curriculum: Separation and identification of the amino acid residues in an unknown dipeptide using FMOC derivatization. J. Chem. Educ. 1999, 76 (6), 820−825. (27) Lagane, B.; Treilhou, M.; Couderc, F. Capillary electrophoresis: theory, teaching approach and separation of oligosaccharides using indirect UV detection. Biochem. Mol. Biol. Educ. 2000, 28 (5), 251− 255. (28) Williams, K. R.; Bhavin, A.; German, I.; Russell, T. Determination of a diffusion coefficient by capillary electrophoresis. J. Chem. Educ. 2002, 79 (12), 1475−1476. (29) Vestling, M. M. A peptide HPLC experiment for biochemistry laboratory. J. Chem. Educ. 1991, 68 (1), 72. (30) Ermakov, S. V.; Zhukov, M. Y.; Capelli, L.; Righetti, P. G. Wall adsorption in capillary electrophoresis: Experimental study and computer simulation. J. Chromatogr. A 1995, 699 (1−2), 297−313. (31) GPMAW home page; http://www.gpmaw.com/ (accessed December 2016). (32) Isoelectric Point Calculator; http://isoelectric.ovh.org/ (accessed December 2016). (33) Swiss Institute of Bioinformatics pI and Molecular Weight Calculator; http://web.expasy.org/compute_pi/ (accessed December 2016).

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Capillary Zone Electrophoresis for the Analysis of Peptides: Fostering

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