Utilization of Lysozyme Charge Ladders to Examine the Effects of

Aug 13, 2009 - Center For Biotechnology and Interdisciplinary Studies ... The ion exchange separation showed a significant degree of variation in the ...
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Utilization of Lysozyme Charge Ladders to Examine the Effects of Protein Surface Charge Distribution on Binding Affinity in Ion Exchange Systems )

Wai Keen Chung,†,‡ Steven T. Evans,†,‡ Alexander S. Freed,†,‡ James J. Keba,§ Zachary C. Baer,†,‡ Kaushal Rege, and Steven M. Cramer*,‡,† Department of Chemical and Biological Engineering and ‡Center For Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, §Process Research and Development, Genentech, South San Francisco, California 94080, and Department of Chemical Engineering, Arizona State University, Tempe, Arizona 85287 )

†

Received June 14, 2009. Revised Manuscript Received July 24, 2009 A lysozyme library was employed to study the effects of protein surface modification on protein retention and to elucidate preferred protein binding orientations for cation exchange chromatography. Acetic anhydride was used as an acetylating agent to modify protein surface lysine residues. Partial acetylation of lysozyme resulted in the formation of a homologous set of modified proteins with varying charge densities and distribution. The resulting protein charge ladder was separated on a cation exchange column, and eluent fractions were subsequently analyzed using capillary zone electrophoresis and direct infusion electrospray ionization mass spectrometry. The ion exchange separation showed a significant degree of variation in the retention time of the different variants. Several fractions contained coelution of variants, some with differing net charge. In addition, several cases were observed where variants with more positive surface charge eluted from the column prior to variants with less positive charge. Enzymatic digest followed by mass spectrometry was performed to determine the sites of acetylation on the surface of the variants eluting in various fractions. Electrostatic potential maps of these variants were then generated to provide further insight into the elution order of the variants.

1. Introduction Ion exchange chromatography is widely employed for a range of biopharmaceutical applications. In order to further optimize and improve ion exchange resins for separation processes, it is essential to gain a better understanding of protein interactions in these systems. Kopaciewicz and co-workers1 have established that charge clusters play an important role in protein retention in ion exchange. Chicz and Regnier2 have shown that alteration of a single amino acid residue can cause significant changes in protein retention behavior if the mutation occurs at a site that interacts with the resin surface. Roush et al.3,4 have used electrostatic potential surfaces and electrostatic interaction free energies to simulate the binding of rat cytochrome b5 to an anion exchange surface. Yao and Lenhoff 5,6 studied the electrostatic interaction free energies for cytochrome c variants binding onto a cation exchanger and modeled the protein retention behavior using energy-minimized 3D models to demonstrate that charge density and distribution play an important role in determining protein binding in ion exchange systems. We have used a homologous library of site-directed mutants of cold shock protein B (CspB) to examine the effects of charge modification on the retention behavior in cation exchange chromatography and to elucidate preferred binding regions on the protein surface.7 *To whom correspondence should be addressed. E-mail: [email protected]. Phone:(518) 276-6198. Fax: (518) 276-4030. (1) Kopaciewicz, W.; Rounds, M. A.; Fausnaugh, J.; Regnier, F. E. J. Chromatogr. 1983, 266, 3–21. (2) Chicz, R. M.; Regnier, F. E. Anal. Chem. 1989, 61(18), 2059–2066. (3) Gill, D. S.; Roush, D. J.; Willson, R. C. J. Chromatogr., A 1994, 684(1), 55–63. (4) Roush, D. J.; Gill, D. S.; Willson, R. C. Biophys. J. 1994, 66(5), 1290–1300. (5) Yao, Y.; Lenhoff, A. M. Anal. Chem. 2004, 76(22), 6743–6752. (6) Yao, Y.; Lenhoff, A. M. Anal. Chem. 2005, 77(7), 2157–2165. (7) Chung, W. K.; Hou, Y.; Freed, A.; Holstein, M.; Makhatadze, G. I.; Cramer, S. M. Biotechnol. Bioeng. 2009, 102(3), 869–881.

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Site-directed mutagenesis or the use of protein variants from different species to obtain a library of homologous proteins enables correlation between protein retention changes and alterations in protein surface properties. However, the generation of the mutant library is a labor intensive and costly procedure. Further, an extensive library of protein mutants would have to be generated to fully evaluate the effects of protein surface modifications on the retention behavior. The use of protein variants from different species is less labor intensive but is limited by the number of variants that are available. Chemical modification of the protein surface offers an easier, faster, and less costly alternative. Gao et al.8 have developed a method of partially acetylating protein molecules to generate protein charge ladders in an attempt to determine the effective charge of a protein in solution by capillary electrophoresis. Protein charge ladders have also been employed in other investigations. Cordova et al.9 examined the effects of coatings on capillary walls on protein adsorption using protein charge ladders. Carbeck et al.10 used protein charge ladders to determine the net charge of the proteins in both liquid and gas phases using capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESIMS). Negin and Carbeck11 developed a method of determining the effects of proton linkage and longrange electrostatic interaction on protein folding using protein charge ladders in capillary electrophoresis. Gitlin et al.12 found (8) Gao, J. M.; Gomez, F. A.; Harter, R.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91(25), 12027–12030. (9) Cordova, E.; Gao, J. M.; Whitesides, G. M. Anal. Chem. 1997, 69(7), 1370– 1379. (10) Carbeck, J. D.; Severs, J. C.; Gao, J. M.; Wu, Q. Y.; Smith, R. D.; Whitesides, G. M. J. Phys. Chem. B 1998, 102(51), 10596–10601. (11) Negin, R. S.; Carbeck, J. D. J. Am. Chem. Soc. 2002, 124(12), 2911–2916. (12) Gitlin, I.; Mayer, M.; Whitesides, G. M. J. Phys. Chem. B 2003, 107(6), 1466–1472.

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that charge compensation which occurred during the generation of protein charge ladders had an effect on the overall change in net charge on the protein surface. Allison et al.13 developed a predictive model of protein electrophoretic mobility using data obtained with protein charge ladders. Piaggio et al.14 used two physicochemical models to examine the hydrodynamic radius and net charge of a protein in capillary electrophoresis using protein charge ladders. Szymanski et al.15 employed lysozyme charge ladders to determine the electrophoretic mobility of the proteins in the presence of a nonionic surfactant and demonstrated that the effective charge of the protein was unaffected by the presence of the surfactant. Brautignan et al.16 carried out chemical modification of surface lysine residues on horse cytochrome c using 4-chloro-3,5-nitrobenzoate to elucidate possible binding regions on the protein surface to a CM-cellulose cation exchanger. Dismer and Hubbuch17 used a dye labeling process with lysozyme bound onto different cation exchangers to elucidate the orientation of lysozyme on various surfaces. Xu et al.18 generated a cation exchange surface that consisted of succinate residues that could covalently bind to surface lysines of horse cytochrome c that came into contact with the surface. The succinate residues were subsequently hydrolyzed from the resin surface and analyzed using tryptic digest mass spectrometry to determine the regions on the protein that came into contact with the resin surface. While interesting, this method requires the use of a specially created cation exchange surface and is not generally applicable to commercial cation exchangers. In this paper, we employ acetic anhydride as a chemical modifier to generate a protein charge ladder based on hen egg white lysozyme. The lysozyme charge ladder is then used as a tool to study the effects of protein surface charge modifications on protein binding affinity and preferred orientation in cation exchange chromatography (CEX). Capillary zone electrophoresis (CZE) is used in concert with direct infusion electrospray ionization mass spectrometry (ESI-MS) to analyze CEX eluent fractions and to determine the types of variants present within each fraction. Enzymatic digest and MS are subsequently performed to determine the sites of acetylation on the protein variant surface. Finally, electrostatic potential maps of these variants are generated to provide further insight into the elution order of the variants.

2. Experimental Methods 2.1. Materials and Reagents. Chicken egg white lysozyme, tris-base, glycine, hydrochloric acid, mono- and dibasic sodium phosphate, sodium chloride, Trypsin, GluC, AspN, ArgC, R-cyano-4-hydroxycinnamic acid, methanol, trifluoroacetic acid (TFA), ammonium bicarbonate, and 1,4-dioxane were purchased from Sigma Chemical Co. (St. Louis, MO). Acetic anhydride, sodium hydroxide, and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ). Ethanol was purchased from Aldrich (Milwaukee, WI). Dithiothreitol was purchased from Promega Corp. (Madison, WI). ZipTips were purchased from Millipore Corp. (Billerica, MA). An amine capillary regeneration solution was purchased from Beckman-Coulter (Fullerton, CA). Strong (13) Allison, S. A.; Carbeck, J. D.; Chen, C. Y.; Burkes, F. J. Phys. Chem. B 2004, 108(14), 4516–4524. (14) Piaggio, M. V.; Peirotti, M. B.; Deiber, J. A. Electrophoresis 2005, 26(17), 3232–3246. (15) Szymanski, J.; Pobozy, E.; Trojanowicz, M.; Wilk, A.; Garstecki, P.; Holyst, R. J. Phys. Chem. B 2007, 111(19), 5503–5510. (16) Brautigan, D. L.; Fergusonmiller, S.; Margoliash, E. J. Biol. Chem. 1978, 253(1), 130–139. (17) Dismer, F.; Hubbuch, J. J. Chromatogr., A 2007, 1149(2), 312–320. (18) Xu, W. S.; Zhou, H.; Regnier, F. E. Anal. Chem. 2003, 75(8), 1931–1940.

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cation-exchange (SCX) columns (sulfopropyl on cross-linked polymethacrylate polyol ester) Protein-Pak SP8HR (8 μm, 100 mm  4.6 mm ID) were a gift from Waters (Milford, MA, USA). 2.2. Equipment. Analytical linear gradient experiments were performed using an Akta explorer 100 chromatography system controlled by the Unicorn 5.0 software. Fractions of the column eluent were collected using an Advantec SF-2120 Super fraction collector. A Beckman PACE MDQ system was used to carry out the capillary electrophoresis experiments. A PC with Windows 95 and Beckman P/ACE System MDQ v 2.3 software was used for data analysis. The capillary used was an eCAP amine capillary with an internal diameter of 75 μm with a separation length of 50.0 cm from the inlet to the detection window. Both eCAP amine capillaries and regeneration solution were purchased from Beckman Coulter (Fullerton, CA). A Thermo LTQ Orbitrap XL linear trap-orbitrap mass spectrometer was used to carry out protein and peptide direct infusion mass spectrometry analysis of selected column eluent fractions and fraction digests. A Bruker Daltonik Ultraflex III MALDI-TOF-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) was used to carry out the peptide mass spectrometry analysis on digests of selected column eluent fractions.

2.3. Procedures. 2.3.1. Protein Charge Ladder Formation. A 10 mg/mL solution of lysozyme was first prepared in DI water. The protein solution was then brought to pH 12.0 using 1.0 N NaOH. A solution of acetic anhydride (100 mM in 1,4dioxane) was prepared by adding 100 μL of acetic anhydride to 10 mL of 1,4-dioxane. 1.5 mL of acetic anhydride was then added to 48.5 mL of the lysozyme solution. After 1 min, the reaction was quenched by lowering the pH of the mixture to 7.0 using 1.0 N HCl. 2.3.2. Cation Exchange Chromatography. A Waters Protein-PAK 8HR SP column was used for the analysis and separation of the lysozyme charge ladder mixture. 1.0 mL of a 10.0 mg/ mL lysozyme charge ladder solution was injected onto the column. Linear gradient elution runs from 100% buffer A (50 mM sodium phosphate, pH 6.0) to 100% buffer B (50 mM sodium phosphate containing 300 mM sodium chloride, pH 6.0) in 35 column volumes at a flow rate of 1.5 mL/min were carried out to obtain retention time data on the variants. The column effluent was monitored at 280 nm and eluent fractions were collected at a rate of 2 fractions per minute. 2.3.3. Capillary Zone Electrophoresis (CZE). Cation exchange eluent fractions were analyzed using capillary zone electrophoresis. Analysis was carried out on a 50 cm Beckman Coulter eCAP amine capillary using 25 mM Tris-192 mM Gly buffer, pH 8.4 and an applied voltage of 20 kV in reverse polarity. Samples were loaded into the capillary by applying a positive pressure of 0.5 psi for 5 s. Detection of all samples was carried out using a photodiode array detector with UV absorbance set at 214 nm. Prior to CZE analysis, eluent fractions were subjected to buffer exchange using a 0.5 mL capacity Millipore Centriplus centrifugal concentrator (YM-3), which has a molecular weight cutoff of 3000 Da in order to remove the salt from the cation exchange analytical step.

2.3.4. Mass Spectrometry of Cation Exchange Fractions. All samples were desalted using Millipore reversed-phase ZipTip pipet tips before mass spectrometry analysis using the vendor’s protocol. The samples were eluted from the ZipTips using an aqueous eluent containing 90% (v/v) acetonitrile and 0.1% (v/v) TFA. Direct infusion electrospray ionization mass spectra were obtained on a Thermo LTQ Orbitrap XL linear trap-orbitrap mass spectrometer. Samples were introduced into the ion source using an autosampler without prior enzymatic digestion delivered at a flow rate of 50 μL/min. The source voltage was set to 3.9 kV and the capillary temperature set to 275 °C. Data was collected in the positive ion mode in a m/z range from 500 to 2000. The mass spectrometer was calibrated and optimized for the detection of the lysozyme charge ladder feed mixture. Instrument control, data Langmuir 2010, 26(2), 759–768

Chung et al. acquisition and data processing were performed using Xcalibur 2.0 software (Thermo). The QualBrowser software was used to smooth, baseline subtract, and extract charge envelope peaks having signal-to-noise ratios greater than 10 to the protein molecular weight range of interest (14 000-15 000 Da).

2.3.5. Enzymatic Digestion of Cation Exchange Fractions. Dithiothreitol (DTT) was added to each fraction to a concentration 4 mM. Elevated temperatures (75 °C for 1 h) were used to denature the protein. The pH was adjusted to 8.0, and the fraction was aliquoted into four equal volumes. Iodoacetamide was added to yield an iodoacetamide:cysteine ratio of 1000:1 and allowed to react for 1 h at room temperature. Enzyme (either TPCK treated trypsin, GluC, AspN, or ArgC purchased from Sigma, St. Louis, MO) was then added to a substrate: enzyme ratio of 30:1 and incubated at 37 °C for 24 h with periodic mixing. The digest was allowed 1 h to cool to room temperature and glacial acetic acid was added to stop the digest by bringing the pH to 3.0. These peptide mixtures were then desalted using ZipTips for mass spectrometry analysis or stored frozen at -80 °C.

2.3.6. Mass Spectrometry Analysis of Digested Cation Exchange Fractions. MALDI-TOF mass spectra were recorded using a Bruker Daltonik Ultraflex III MALDI-TOF-TOF mass spectrometer. Selected CEX column eluent fractions that were digested using the four enzymes mentioned above were desalted and spotted onto a target plate using ZipTips. Each fraction was analyzed using a separate spot that was digested with one of the four enzymes. The matrix used was R-cyano-4-hydroxycinnamic acid and ionization was performed using a Nd:YAG “smart” laser beam. Calibration of the mass range in reflectron mode was performed using a Peptide Calibration Standard 2 mixture (Bruker Daltonik, part #222570) and using the peptides generated from the tryptic digest of the charge ladder mixture. Direct infusion electrospray ionization mass spectra were obtained on a Thermo LTQ Orbitrap XL linear trap-orbitrap mass spectrometer. Selected CEX column eluent fractions that were digested using the four enzymes were desalted using ZipTips and introduced into the ion source using an autosampler at a flow rate of 50 μL/min. Data was collected in the positive ion modes in a mass range from m/z of 400 to 2000 Da. The mass spectrometer was calibrated and optimized for the detection of the lysozyme charge ladder feed mixture peptides. Instrument control, data acquisition, and data processing were performed using Xcalibur 2.0 software (Thermo). The QualBrowser software was used to smooth, baseline subtract, and extract charge envelope peaks having signal-to-noise ratios greater than 5 to the peptide m/z range of interest (400-2800 Da).

2.3.7. Charge Ladder Variant Electrostatic Potential Surface Analysis. To study the distribution of electrostatic potential near the protein, a series of Poisson-Boltzmann calculations were preformed on the native protein and acetylated variants. Each variant was developed by manually acetylating the native structure of the desired lysine. The modified protein structure was then minimized using the AMBER 94 force field. The proteins were then parametrized with the PARSE force field due to its ability to properly account for implicit solvent effects. The modified lysines were then assigned charges based on known PARSE parameters for similar chemical structures. The Adaptive Poisson-Boltzmann Solver (APBS)19-22 package was used to generate electrostatic distributions using a linear Poisson-Boltzmann solution under 0.15 M ionic concentration. Electrostatic potential maps were depicted using the VMD molecular graphics viewers.23

(19) Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98(18), 10037–10041. (20) Holst, M.; Saied, F. J. Comput. Chem. 1993, 14(1), 105–113. (21) Holst, M. J.; Saied, F. J. Comput. Chem. 1995, 16(3), 337–364. (22) Bank, R. E.; Holst, M. Siam Journal on Scientific Computing 2000, 22(4), 1411–1443. (23) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14(1), 33–38.

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3. Results and Discussion 3.1. Cation Exchange Chromatography. The lysozyme charge ladder mixture was generated as described in the experimental section using the approach of Gao et al.8 The addition of acetic anhydride caused the acetylation of the ε-amino group of the six surface lysines as well as the N-terminal R-amino group. The acetylation process allowed for the modification of the protein surface charge with negligible effects on the physical properties of the protein due to the small molecular weight increase brought about by the reaction. Through partial acetylation of lysozyme, a homologous mixture of protein variants with varying charge density and distribution, was generated. The resulting charge ladder was then separated by cation exchange chromatography (CEX) using a 35 column volume NaCl linear gradient. Figure 1 shows the resulting chromatogram obtained during the CEX run. As can be seen in the figure, a large number (∼40) of peaks were observed under these chromatographic conditions. While these chromatography experiments were carried out with the Waters Protein-PAK SP 8HR resin, it is expected that significant variation in retention times would also be observed for this protein library on other CEX resin systems. Acetylation of a surface lysine residue reduces the net charge of the protein by one unit. On the cation exchange column, variants possessing more positive charge (i.e., less acetylated variants) were retained longer as compared to molecules with less positive charge (i.e., more acetylated variants). Native lysozyme, having more surface positive charge than any other variant, was retained the longest in the column and eluted at approximately 128 min. Lysozyme has six surface lysine residues and an N-terminal R-amino group, which are susceptible to acetylation. It has been reported by Suckau et al.24 that the lysine residues and the N-terminal amino group in hen egg white lysozyme have a much higher reactivity as compared to the surface histidine and tyrosine residues. Acetylation of these additional amino acids only occurs under conditions where the amount of acetic anhydride is in large excess (10 000-fold molar excess). It was therefore assumed that only the lysines and N-terminal amino groups were acetylated. Acetylation of the histidines and tyrosines, if present, would occur only to a marginal extent and were not detected by our analyses. Given the combinations possible through partial acetylation of these 7 surface amino groups there could be up to 128 different variants present within the lysozyme charge ladder mixture. However, as shown in Figure 1, there are significantly fewer peaks in the chromatogram, indicating the likelihood of variant coelution. Fractions from the column effluent during the gradient run were then evaluated using CZE and direct infusion ESI-MS in order to determine the identity of the variants present in the peaks shown in Figure 1. 3.2. Capillary Electrophoresis Analysis. As mentioned in the Introduction, lysozyme charge ladders were developed by Gao et al.8 to determine the effective charge of protein molecules in solution by capillary electrophoresis. Depending on the degree of acetylation, the reduction in electrophoretic mobility of the protein gives rise to different electropherogram peaks or “rungs” when a protein charge ladder mixture separated by CZE. Since lysozyme has six surface lysines, six variant peaks were obtained in CZE analyses of their charge ladders.8 It has also been reported by Gao et al.25 that since acetylation of the N-terminal R-amino group reduces the net charge of the protein by less than a singular unit of charge, N-terminal amino acetylated variants do not elute (24) Suckau, D.; Mak, M.; Przybylski, M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(12), 5630–5634. (25) Gao, J. M.; Whitesides, G. M. Anal. Chem. 1997, 69(4), 575–580.

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Figure 1. Chromatogram showing the separation of a lysozyme charge ladder mixture on a Waters Protein-PAK SP8HR cation exchange column.

Figure 2. Electropherogram using a Beckman Coulter Amine capillary (Separation voltage: 20 kV, Reverse polarity): (a) lysozyme charge ladder mixture, (b) CEX fraction 44, (c) CEX fraction 178, (d) CEX fraction 184.

as a discrete peak, but can cause peak splitting to occur in the electropherogram. Figure 2a is an electropherogram of a lysozyme charge ladder mixture that was separated by CZE. V(N) is used to denote the number of acetylated lysines. For example, V5 refers to a variant with 5 acetylated amino groups. The charge ladder mixture was separated using a positive capillary with the electrodes in a reverse polarity setting. This resulted in the elution of highly acetylated variants before less acetylated variants, followed by the native 762 DOI: 10.1021/la902135t

protein. The peak splitting for V6 and V5 was a result of the inability of the CZE technique to separate variants that possess N-terminal amino acetylations from those that do not. Thiourea was added to all samples as an electroosmotic flow (EOF) marker. The difference in retention time between each charge ladder peak and the EOF marker was then used to identify the variants present in the CEX eluent fractions during CZE analysis. The general trend observed during the CZE analysis of the cation exchange eluent fractions was that highly acetylated Langmuir 2010, 26(2), 759–768

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Figure 3. Protein analysis using direct infusion ESI-MS (a) lysozyme charge ladder mixture, (b) CEX fraction 44, (c) CEX fraction 178, (d) CEX fraction 184.

variants were found in the earlier eluent fractions while the less acetylated variants were present in the later fractions. This would be expected as the highly acetylated variants possessed less positive surface charge and would expected to be less strongly retained on the cation exchanger as compared to less acetylated variants or native lysozyme. However, at many points along the elution train, unique retention behavior was observed which ran contrary to the expected trends. For example, the electropherogram from the analysis of CEX eluent fraction 44 (Figure 2b) shows that variants 5 and 4 coeluted in this fraction. This is unexpected since the difference in protein surface net charge should have resulted in the two variants eluting at different points of the gradient. It was also interesting to note that, at several points along the CEX gradient, less acetylated variants eluted from the cation exchanger before highly acetylated variants. An example of this is given in the electropherograms of CEX eluent fractions 178 and 184, shown in in Figure 2c,d, respectively. V1 was found in CEX eluent fraction 178, while V2 was found in fraction 184. It would be expected that variants with more positive surface charge (i.e., less acetylated) would be retained more strongly on the cation exchanger as compared to variants with less surface positive charge (i.e., more acetylated). However, as can be seen in this figure, a reversal in the elution order occurred. 3.3. Mass Spectrometry Analysis of Protein Fractions. CZE enables rapid determination of the number of acetylated lysine groups on the surface of the protein. However, this technique is limited by its detection sensitivity. Mass spectrometry on the other hand has a much higher sensitivity (femtomole Langmuir 2010, 26(2), 759–768

sensitivity) allowing for the detection of dilute variants.26 As mentioned above, CZE cannot distinguish two variants if the only difference between them was an acetylation of the N-terminal amino group. In contrast, MS utilizes the increase in molecular weight of the variant to determine the number of acetylated sites on the protein surface, regardless of whether modification occurred at the N-terminal amino group or lysine side chains. Hence, using a combination of the variant CZE elution time with that of the MS spectra, N-terminal amino acetylation could be detected. This was important, as the separation on the cation exchanger was performed at pH 6 where the N-terminal group would be charged and has an impact on the retention of the protein on the cation exchanger. Acetylation of a lysine residue increases the molecular weight of the protein by 42. Therefore, by looking at the increase in molecular weight by increments of 42 when compared with the molecular weight of native lysozyme (MW 14 305), the number of acetylated sites on the protein surface can be determined. As described in the Experimental Section, the lysozyme charge ladder mixture was desalted and injected into the mass spectrometer without further treatment. The resulting mass spectra of the feed mixture (Figure 3a) produced 8 peaks due to varying degrees of acteylation of the 6 lysine and N terminal amino groups on lysozyme. This is in contrast to the CZE analysis of the feed, which produced only 7 lysozyme variant or native peaks. Figure 3b,c,d shows the mass spectra of CEX eluent fractions (26) Han, X. M.; Aslanian, A.; Yates, J. R. Curr. Opin. Chem. Biol. 2008, 12(5), 483–490.

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Figure 4. Graphical depiction of the possible types of acetylated variants within each eluent fraction.

44, 178, and 184, respectively. The number of acetylated sites on each variant in the 3 fractions obtained from direct infusion ESI MS agreed well with CZE analysis of these fractions, indicating that the acetylation had occurred on the lysine side chains and not the N-terminal amino group for the variants within these fractions. Using a combination of CZE and direct infusion ESI MS as orthogonal analytical techniques, the identity of the variant types within each cation exchange eluent fraction can be successfully determined. Figure 4 shows the variant assignments for all of the CEX eluent fractions and depicts the elution order of the variants from the cation exchange separation step. As can be seen in the figure, the overall trend showed elution of variants with less positive surface charge (i.e., more acetylated) before variants with more positive surface charge (i.e., less acetylated) with the native protein being retained the longest. In addition, multiple cases of coelution and elution order reversal were observed. In order to study the changes in protein retention behavior with respect to the site of acetylation, enzymatic digest mass spectrometry was performed. 3.4. Determination of Variant Site Modification using Mass Spectrometry. Four different enzyme digests were performed with selected cation exchange eluent fractions within each peak on the CEX chromatogram. The use of four restriction digest enzymes ensured sufficient coverage of all potential modified protein sites. The digested peptides were subjected to both MALDI-TOF MS and direct infusion ESI-MS analysis for peptide mass fingerprinting. Mass spectrometry analyses have been previously performed using single enzyme treatment to identify sites of modification after fluorescently labeling lysozyme.17,24 A representative result in shown in Figure 5, which presents the mass spectra of a tryptic digested native lysozyme sample and CEX eluent fraction 232. In this figure, all of the peptides resulting from the tryptic digests are shown along with their mass to charge ratio and the assigned amino acid sequence (in parentheses). For the native lysozyme (Figure 5a), a peptide fragment with a mass of 1805.1 Da was seen, which corresponded to a peptide fragment with an unacetylated lysine at position 97. In contrast, in eluent fraction 232 (Figure 5b), the peptide mass fragment of 1805.1 Da was absent, while a new peptide fragment with a mass of 1847.1 Da appeared (note: this corresponds to the molecular weight of the fragment with an acetylated lysine at position 97). Using a combination of mass spectra data with the different restriction enzymes, fraction 232 was found to contain only a single variant 1 with an acetylated lysine at position 97. This multienzyme digest mass spectrometry approach was employed to evaluate a number of fractions from the cation 764 DOI: 10.1021/la902135t

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exchange experiment to explicitly identify the modification sites of the variants. Table 1(a) is an abbreviated list of peptides used in the determination of the acetylation at a particular lysine site on the protein surface. This table includes the name of each peptide (label), the mass, the amino acid residues from the protein, the enzyme employed to generate the peptide (note: in some cases two different enzymes resulted in the same peptide), and the actual sequence of the peptide. Labels with the superscript N correspond to the native protein, and those with superscript A correspond to acetylated peptides. Specific acetylation sites are indicated in the sequence using the subscript AC. All cysteine thiols were capped using iodoacetamide and are indicated with subscript M. Table 1(b) shows the assignment of the variants that were determined with a high degree of confidence from the presence and loss of various peaks from the mass spectra. This table presents the fraction time from the CEX gradient, the variant assignment (from the CZE and direct infusion MS analyses performed above), the specific acetylation sites, and peptides that were absent and those that appeared relative to the native lysozyme (note: the peptides were defined in Table 1a). As can be seen in the table, 15 variants were able to be identified from the gradient fractions using this approach. The identified peptides and the corresponding specific acetylation sites provide the exact sites of modification responsible for the changes of protein retention behavior observed in the CEX experiments. Further, this identification of specific sites of modification corroborates the variant identifications made through CZE and direct infusion ESI (e.g., V2 in fraction 92 has only 2 acetylation sites at positions 13 and 97). In addition, these data support some of the unexpected elution trend reversals where less acetylated variants eluted from the cation exchanger before more highly acetylated variants (e.g., V1s in fraction 89 eluting before V2 in fraction 92). The advantage of using this multienzyme digest MS approach is illustrated by the detection of coeluting variant 1s in fraction 89. Both variants are inseparable by CZE or direct infusion MS due to similarities in molecular weight and net surface charge. However, analysis of the fraction 89 peptide fragments indicated the presense of peptides with the Lys 1 side chain acetylated (peptide 1A1 in Table 1a observed) as well as peptides with the N-terminal amino group acetylated (peptide 5N did not disappear from the spectra indicating N-terminal modification). Since only variant 1s were present in this fraction, the combined analysis identifies the two variant 1s in this fraction. There are several reasons we were limited in the number of specific variants that could be identified. These include sensitivity of the mass spectrometry for a given peptide, the relative concentration of a given variant due to differences in reacitity during the acetylation, and the presence of multiple variants in a single fraction. When performing mass spectrometry, the inability to detect a given peak does not imply its absence in the sample, since the presence of other peptides may mask its detection. In addition, it has been reported in the literature that the lysozyme amino groups have different reactivities during acetylation.24 This can result in some variants being present at relatively low concentration, making their detection difficult. Coelution of multiple variants of the same type (e.g., variant 2’s) in a single fraction could pose a problem in the identification of specific acetylation sites when the number of sites identified by enzymatic digest-MS exceeds the number expected for that variant (e.g., 3 or more modifications for V2). This problem also extends to cases where coelution of multiple variants of different types (e.g., variants 2 and 3) occurs in a single fraction. Accordingly, we focused our analysis on fractions that contained a single variant. Langmuir 2010, 26(2), 759–768

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Figure 5. Mass spectra of Trytic digested (a) native lysozyme; (b) CEX fraction 232.

To further investigate the importance of the lysine residues identified through this multienzyme digest mass spectrometry approach, electrostatic potential maps were generated for native lysozyme, all of the identified variant 1s, and the variant 2 in fraction 92. 3.5. Examination of the Electrostatic Potential Maps of Native and Acetylated Lysozyme. It has been previously reported that the electrostatic potential (EP) is a major determinant in protein retention on ion exchange surfaces.7 Accordingly, it is instructive to examine the EP of the native protein and specific acetylated variants to determine the effect of acetylation on the EP of the variants. As a basis for comparison, a study of the change in EP between native lysozyme and all of the variant 1s identified by enzyme digest-MS was carried out. All variant 1s will have the same surface net charge but varying charge distribution. If surface net charge was the sole determining factor in protein retention, all variant 1s would coelute; however, clearly Langmuir 2010, 26(2), 759–768

this was not the case as seen in Table 1b. The elution order of the identified variant 1s with respect to their site of acetylation was positions 1 and the N-terminal, 33, and 97. As expected, all of these variant 1s exhibited weaker retention than the native lysozyme. Figure 6a shows the front and rear views of the EP isosurface for native lysozyme. The blue isosurface depicts a level of þ1 kT/e, while the red isosurface depicts a level of -1 kT/e. As seen in the figure, the N-terminus amino group, Lys 1, and Lys 33, are located within large positive clusters on the protein surface. Thus, the loss of a positive charge at these three locations would be expected to have a significant impact on the binding affinity to the resin since these charge clusters could comprise a preferred binding region for the native protein. This binding region (indicated by the white dotted area A in Figure 6a comprises the N-terminus amino group, Lys 1, Arg 5, Lys 33, Arg 114, and Arg 128. This can be seen by the localized loss of positive EP shown in Figure 6b,c,d. DOI: 10.1021/la902135t

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Table 1. (a) Abbreviated List of Peptide Fragments Used in the Identification of the Sites of Acetylation on the Surface of Lysozyme; (b) List of Variants Identified from the Lysozyme Charge Ladder Mixture Using Enzymatic Digest Mass Spectrometrya (a) Abbreviated List of Relevant Mass Spectrum Peaks from the Enzymatic Digest of Lysozyme label

peptide mass

residues

enzyme(s)

sequence

1N 2N 3N 4N 5N 6N 7N

606.7 1049.5 1334.5 1506.7 1509.8 1805.1 2739.0

1-5 6-14 115-125 88-101 2-14 97-112 22-45

ArgC/Trypsin Trypsin ArgC/Trypsin AspN/GluC Trypsin Trypsin Trypsin

KVFGR CmELAAAMKR CmKGTDVQAWIR ITASVNCmAKKIVSD VFGRCMELAAAMKR KIVSDGNGMNAWVAWR GYSLGNWVCmAAKFESNFNTQATNR

1A1 1A2 2A 3A 4A1 4A1 4A2 5A 6A 7A

648.8 690.4 1092.3 1376.6 1548.8 1548.8 1590.8 1551.8 1847.1 2779.0

1-5 1-5 6-14 115-125 88-101 88-101 88-101 2-14 97-112 22-45

ArgC/Trypsin ArgC/Trypsin Trypsin ArgC/Trypsin AspN/GluC AspN/GluC AspN/GluC Trypsin Trypsin Trypsin

KAcVFGR (Lys or N-terminal modified) KAcVFGR (both modified) CmELAAAMKAcR CmKAcGTDVQAWIR ITASVNCmAKAcKIVSD ITASVNCmAKKAcIVSD ITASVNCmAKAcKAcIVSD VFGRCMELAAAMKAcR KAcIVSDGNGMNAWVAWR GYSLGNWVCmAAKAcFESNFNTWSTNR

(b) Lysozyme Charge Ladder Variant Assignments for Selected Fractions fraction time (min) assignment

a

sites of acetylation

peptides absent upon acetylation peptides observed upon acetylation

V7 NTerm, Lys1, Lys13, Lys33, lys96, Lys97, Lys116 V6 Lys1, Lys13, Lys33, lys96, Lys97, Lys116 V5 Lys1, Lys13, Lys33, Lys97, Lys116 V5 Lys1, Lys13, Lys33, lys96, Lys97 V3 NTerm, Lys33, Lys97 V2 Lys33, Lys97 V2 Lys1, Lys97 V2 Lys97, Lys116 V2 Lys13, Lys33 V1 NTerm or Lys1 V2 Lys13, Lys97 V1 Lys33 V1 Lys97 Native a Identified from direct infusion ESI MS.

6 9 13 20 47 72 75 79 84 89 92 96 116 127

By examining the experimental elution order in concert with the EP maps of the variants, it is clear that the N-terminus amino group, Lys 1, and Lys 33 play an important role in determining the binding of lysozyme on the cation exchange surface. The significance of this region on the lysozyme surface for binding to ion exchange is also supported by other sources in the literature.17 In contrast, Lys 97 is adjacent to negative charge clusters that may interfere with the electrostatic attraction between Lys 97 and the resin surface. Thus, acetylation of this lysine residue would be expected to have less of an impact on the binding affinity to the resin, since it is less likely to be part of a preferred binding region. The effect of this mutation on the EP of the rear face of the protein is shown in Figure 6e. Acetylation of Lys 1 or the N-terminus amino group caused a larger drop in protein retention as compared to acetylating Lys 33 (Table 1b). One possible explanation is that both the N-terminus amino group and Lys 1 are also part of a separate binding region of the protein surface located on the left side of the protein. The second binding region (outlined by the yellow dotted area B in Figure 6a) consists of the N-terminus amino group, Lys 1, Arg 14, His 15, and Arg 68. Since both the N-terminus amino group and Lys 1 are located at the interface of binding regions A and B, loss of a positive charge at these two positions could have a larger impact on protein retention as compared to Lys 33, which is only involved in one binding face of the protein. In addition, since these two residues are in close proximity and part of the 766 DOI: 10.1021/la902135t

1N - 7N 1N - 7N 1N - 7N 1N, 2N, 4N - 7N 1N, 4N, 6N, 7N 4N, 6N, 7N 1N, 4N - 6N 3N, 4N, 6N 2N, 5N, 7N 1N 2N, 4N, 6N 7N 4N, 6N 1N - 7N

1A2, 2A, 4A, 5A2, 7N, 8N 1A1, 2A, 4A, 5A2, 8N 1A1, 2A, 4A, 5A1, 7A, 8A 1A1, 2A, 4A2, 7A 1A1, 4A1, 6A, 7A 4A1, 6A, 7A 1A1, 4A1, 6A 3A, 4A1, 6A 2A, 5A, 7A 1A1 2A, 4A1 - 6A 7A 4A1, 6A -

same binding regions, loss of charge at either position would be expected to have the same impact on protein retention as is shown by the coelution of the two variant 1s in fraction 89 of Table 1b. The importance of the Lys 1 and the N-terminus amino group as part of the preferred binding regions on the protein surface is further highlighted by the fact that the loss of positive charge at either position caused a larger drop in protein retention than acetylating two residues at other positions on the protein surface. From the list in Table 1b, variant 2 with Lys 13 and Lys 97 acetylated had less of an impact on the protein retention time than acetylating either Lys 1 or the N-terminus amino group. The EP map of this variant is shown in Figure 7. As described above, lysine 97 is not expected to be part of any preferred binding region. On the other hand, lysine 13 is located on the periphery of binding region B at the right side of the protein shown in Figure 6e. Loss of the positive charge at position 13 would alter variant retention time, but since the EP on the front face of the protein is unaffected (Figure 7, front), the drop in retention time would not be as significant as compared to acetylating Lys 1 or the N-terminus amino group (Figure 6b,c). When comparing binding regions, it is also important to consider the underlying charges that contribute to these binding regions as well as the size of the interaction surface. For example, as shown in Figure 6a, while region B has 5 underlying charges (N-terminus amino group, Lys 1, Arg 14, His 15, and Arg 68), region A has six charges (N-terminus amino group, Lys 1, Arg 5, Langmuir 2010, 26(2), 759–768

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Figure 6. Electrostatic potential surface representations of (a) native lysozyme; (b) variant 1 (acetylated N-terminus amino group); (c) variant 1 (acetylated lysine 1); (d) variant 1 (acetylated lysine 33); (e) variant 1 (acetylated lysine 97).

Figure 7. Electrostatic potential surface representations of variant 2 (acetylated lysine 13 and 97).

Lys 33, Arg 114, and Arg 128). In addition, region B is more spread out on the surface as compared to region A. Thus, the charge density of region A will be greater, and this must also be considered when examining the relative binding affinities of these regions and the relative affinities of corresponding variants.

4. Conclusions The lysozyme charge ladder employed in this work enables evaluation of the binding affinities of a relatively large Langmuir 2010, 26(2), 759–768

homologous library of variants for cation exchange chromatography. The results indicated that significant variations in protein retention times were seen as a result of protein surface modifications. CZE and direct infusion ESI MS showed that, at many points in the elution train, variants with different surface net charge coeluted from the CEX surface. It was also observed that, in some cases, variants with more positive surface charge were occasionally eluted before variants with less positive surface charge. Enzyme digest MS was used to determine the exact sites of modification on the surface of the variants and was successful in identifying the modification sites for 15 variants from the charge ladder. The remaining variants were either too difficult to identify using the existing analytical methods or present in concentrations below the limits of detection. Nevertheless, the 15 identified variants were able to provide significant insight into the changes in retention behavior of lysozyme upon acetylation of the protein surface. By examining the changes in EP of the native lysozyme and the identified variants, preferred binding sites on the surface of the protein were identified. These results corroborated that charge distribution and more specifically charge patches played an important role in protein retention. In addition, the data was employed to provide an understanding of the relative elution orders of several variants 1 and 2. In order to obtain more information using this approach, future efforts in ion exchange will use alternative analytical techniques to identify more variants DOI: 10.1021/la902135t

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in the gradient elution fractions. Since the retention time will be an ensemble average of the occupancy time at each particular orientation and since resin properties (e.g., charge distribution, density, and chemistry) will have a major effect on the behavior of these systems, future modeling work using coarse-grained and molecular dynamic simulations will be carried out. Finally, this

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approach of using homologous chemically modified protein libraries will also be used to study other chromatographic systems such multimodal systems. Acknowledgment. This work was supported by National Science Foundation grant CBET 0418413.

Langmuir 2010, 26(2), 759–768