Effect of Separation Temperature on Structure Specific Glycan

Letter pubs.acs.org/ac

Effect of Separation Temperature on Structure Specific Glycan Migration in Capillary Electrophoresis Andras Guttman,*,†,‡ Marta Kerekgyarto,† and Gabor Jarvas†,‡ †

Horváth Csaba Laboratory of Bioseparation Sciences, MMKK, University of Debrecen, Debrecen, Egyetem tér 1, 4032 Hungary MTA-PE Translational Glycomics Research Group, MUKKI, University of Pannonia, Veszprem, Egyetem u. 10, H-8200 Hungary

‡

ABSTRACT: Temperature dependent differential migration shifts were studied in capillary electrophoresis between linear (maltooligosaccharides) and branched (sialylated, neutral and core fucosylated biantennary IgG glycans) carbohydrates. Background electrolytes without as well as with low and high molecular weight additives (ethylene glycol, linear polyacrylamide and poly(ethylene oxide)) were investigated for this phenomena in the temperature range of 20−50 °C. Glucose unit (GU) value shifts were observed with increasing temperature for the all IgG glycans both in additivefree and additive-containing background electrolytes, emphasizing the importance of tight temperature control during glycosylation analysis by capillary electrophoresis. The activation energy concept was applied to understand the structure specific electrophoretic migration of the different sugar molecules. Activation energy values were derived from the slopes of the Arrhenius plots of logarithmic mobility vs reciprocal absolute temperature and compared for the linear and branched sugars as well as for the various background electrolyte additives.

T

where x and t are the distance and time increments. The translational friction coefficient ( f), is considered to be proportional to the viscosity (η) of the background electrolyte,12 and constant c is influenced by molecular configuration13

he emergence of glycoprotein therapeutics in the biopharmaceutical field and recent developments in glycomics-based biomarker discovery call for better understanding of this highly abundant post-translational modification using high-resolution bioanalytical techniques.1,2 The challenging analytical problem of characterizing complex carbohydrates has been addressed by multiple approaches, including nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS), liquid chromatography (LC), and capillary electrophoresis (CE), or the hyphenation of those.3−8 Capillary electrophoresis separates sugars based on their hydrodynamic volume to charge ratios,9 readily recognizing differences in the shape of the glycan structures. In CE, carbohydrates are usually labeled via reductive amination with 8-aminopyrene-1,3,6trisulfonic acid (APTS) that provides excellent fluorescent characteristics and the necessary charge to support the electromigration process.10 Glycan structural annotation is most frequently based on prior use of linear oligosaccharide ladders as retention index to identify branched oligosaccharides.11

f = cη

Electrophoretic mobility (μ) is defined as the electric field normalized electromigration velocity (v/E) and expressed based on eqs 1 and 2 as μ=

η = A e Ea / RT

μ=

(4)

Q e−Ea / RT const

(5)

where const represents a collection of constants including c and A from eqs 3 and 4. Received: October 3, 2015 Accepted: November 6, 2015 Published: November 6, 2015

(1) © 2015 American Chemical Society

(3)

where A is the pre-exponential factor, Ea is the activation energy of the viscous flow, and R is the universal gas constant. Thus, the electrophoretic mobility of a solute ion can be expressed by combining eqs 3 and 4,

THEORETICAL CONSIDERATIONS In electric field-mediated separation methods, when a uniform electric field (E) is applied to a solute molecule (net charge of Q), an electrical force acts on the ion, opposed by a frictional force. When the two forces are counterbalanced, the analyte ions migrate with a steady state velocity (v), Q dx =E dt f

Q cη

According to the Eyring−Polanyi equation,14 the viscosity of the separation medium can be defined as

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v=

(2)

11630

DOI: 10.1021/acs.analchem.5b03727 Anal. Chem. 2015, 87, 11630−11634

Letter

Analytical Chemistry

Figure 1. CE−LIF separations of APTS labeled linear maltooligosaccharides (upper traces) and branched IgG glycans (lower traces) at 20 °C (panel A) and 50 °C (panel B). Peaks correspond to the abbreviated structures listed in Table 1. Background electrolyte, 25 mM lithium acetate (pH 4.75); capillary length, 31 cm (21 cm effective); applied electric field strength, 400 V/cm; injection, 0.5 psi/5 s.

beads. N-Glycan nomenclature was adapted from Harvey et al.18 Capillary Gel Electrophoresis Separation. The PA 800 Plus Pharmaceutical Analysis System, equipped with a solid state laser based fluorescent detector (λex 488 nm and λem 520 nm) was used for all capillary electrophoresis analyses (SCIEX). The separations were accomplished in a 21 cm effective length N−CHO capillary (total length 31 cm, 360 μm o.d., 50 μm i.d., SCIEX) with ±0.1 °C temperature control by the liquid cooling system of the CE instrument. Each sample was analyzed at 20, 30, 40, and 50 °C, using the corresponding buffer systems. All separations were accomplished by applying 12 400 V with the cathode at the injection side (reversed polarity). The background electrolyte was 25 mM lithium acetate (pH 4.75) containing (i) no additive, (ii) 10% ethylene glycol, (iii) 2% linear polyacrylamide (10 kDa), and (iv) 0.4% poly(ethylene oxide) (300 kDa). Samples were pressure injected at 0.5 psi for 5.0 s. For migration time correction and quantification purposes, APTS-labeled maltose (G2) was coinjected with each sample as internal standard. GU values were calculated as described in http://lendulet.uni-pannon.hu/ index.php/en/glystrdb, using the fifth order polynomial approach.19 All runs were done in triplicates, and the 32 Karat, ver 9.1 software package (SCIEX) was used for data acquisition and analysis. Hydrodynamic volumes of relevant maltooligosaccharides (DP 7-12) and major IgG glycans (FA2, FA2[6]G1, FA2[3]G1, and FA2G2) were calculated using TURBOMOLE 6.3 quantum chemical program package and COSMOtherm suite (COSMOlogic GmbH, Leverkusen, Germany).

As eq 5 suggests, the electrophoretic mobility is influenced by differences in activation energy requirement, i.e., the energy required by the solute molecule to overcome the obstacles created by the separation medium.15 In practice, the activation energy values are usually derived from the slopes of the Arrhenius plots16 of logarithmic electrophoretic mobility vs reciprocal absolute temperature (logarithmic version of eq 5). In this paper, the effect of separation temperature was investigated on the differential electromigration shifts between linear and branched carbohydrates in narrow bore capillaries in the range of 20−50 °C. The activation energy values were used to understand the electrophoretic mobility shifts between the different structure sugar oligomers at different temperatures.

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EXPERIMENTAL SECTION Chemicals and Reagents. Human IgG, ethylene glycol, maltose, poly(ethylene oxide) (300 kDa), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). PNGase F was from ProZyme (Hayward, CA). The linear polyacrylamide solution (50%, 10 kDa) was from Polysciences (Warrington, PA). High purity 8-aminopyrene1,3,6-trisulfonic acid (APTS) and the maltooligosaccharide ladder were from SCIEX (Brea, CA). All background electrolyte solutions were filtered through 0.45 μm pore size Acrodisc (Millipore, Billerica, MA) syringe filters and carefully degassed before use. Sample Preparation. In total, 100 μg of IgG was dissolved in 10 μL of 20 mM sodium-bicarbonate solution (pH 7.0), reduced with 1 μL of 50 mM dithiothreitol for 15 min at 65 °C, and alkylated with 1 μL of 50 mM iodoacetamide for 30 min at 37 °C in the dark. For the release of the N-linked glycans, 1 μL of 1 U/μL recombinant PNGase F was added to the samples and incubated for 1 h at 50 °C. The released N-linked glycans were partitioned by CleanSeq magnetic beads (Beckman Coulter, Indianapolis, IN) as described earlier.17 The purified IgG sugars, the maltooligosaccharide ladder, and the maltose internal standard were all fluorescently labeled by the addition of 6.0 μL of 20 mM APTS in 15% v/v acetic acid and 2.0 μL of 1 M sodium cyanoborohydride (in THF) and incubated for 1 h at 50 °C. After the labeling reaction, the tagged glycans were purified again by the CleanSeq (Beckman Coulter) magnetic

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RESULTS AND DISCUSSION In this work, the effect of temperature was investigated on the migration behavior of linear (maltooligosaccharides) and branched (sialylated, neutral, and core fucosylated biantennary IgG glycans) carbohydrates using capillary electrophoresis in the temperature range of 20−50 °C. The activation energy concept was applied to shed light on the electromigration shifts between linear and branched glycans with special respect of the temperature-dependent glucose unit (GU) value changes. The activation energy values were derived from the Arrhenius plots (eq 5) and compared for the various background electrolytes. 11631


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Table 1. Glucose Unit Value Shifts As a Function of Separation Temperature Using 25 mM Lithium Acetate (pH 4.75) Background Electrolytes without and with Monomeric (10% Ethylene Glycol) and Polymeric (2% LPA and 0.4% PEO) Additivesa GU values peak no. 1 2 3 4 5 6 7 8 9 10

a

sample ID

FA21G2S2 FA2[3]G1S1 FA2G2S1 FA2 FA2B FA2[6]G1 FA[3]G1 A2BG2 FA2G2 FA2BG2 average GU values

25 mM Li-acetate 20 °C

30 °C

40 °C

50 °C

5.22 6.31 7.07 8.14 8.65 9.27 9.61 10.03 10.71 11.02

4.95 5.91 6.58 7.50 7.97 8.53 8.85 9.25 9.82 10.12

4.86 5.75 6.34 7.17 7.62 8.11 8.40 8.79 9.32 9.61

4.95 5.83 6.44 7.28 7.74 8.23 8.54 8.93 9.45 9.77

25 mM Li-acetate + 10% EG RSD%

3.12 4.23 4.90 5.74 5.78 6.12 6.10 5.98 6.37 6.25 5.46 25 mM Li-acetate + 2% LPA

Δ

20 °C

30 °C

40 °C

50 °C

−0.27 −0.48 −0.63 −0.86 −0.91 −1.04 −1.07 −1.10 −1.26 −1.26 −0.89

6.29 6.48 7.00 8.00 8.50 9.07 9.38 9.81 10.41 10.73

6.29 6.50 7.01 7.99 8.50 9.04 9.35 9.78 10.37 10.70

6.20 6.41 6.89 7.82 8.33 8.85 9.16 9.59 10.15 10.49

6.09 6.30 6.76 7.62 8.11 8.61 8.91 9.33 9.86 10.19

RSD%

1.55 1.41 1.66 2.24 2.21 2.41 2.34 2.28 2.48 2.34 2.09 25 mM Li-acetate + 0.4% PEO

Δ −0.20 −0.18 −0.24 −0.37 −0.39 −0.47 −0.47 −0.48 −0.55 −0.53 −0.39

peak no.

sample ID

20 °C

30 °C

40 °C

50 °C

RSD%

Δ

20 °C

30 °C

40 °C

50 °C

RSD%

Δ

1 2 3 4 5 6 7 8 9 10

FA21G2S2 FA2[3]G1S1 FA2G2S1 FA2 FA2B FA2[6]G1 FA[3]G1 A2BG2 FA2G2 FA2BG2 average

4.98 5.94 6.72 7.57 8.02 8.67 9.03 9.43 10.16 10.42

5.14 6.13 6.91 7.67 8.20 8.85 9.20 9.63 10.39 10.66

5.25 6.23 7.01 7.79 8.35 8.92 9.27 9.71 10.40 10.72

5.26 6.22 7.00 7.76 8.32 8.88 9.21 9.64 10.30 10.65

2.49 2.18 1.94 1.30 1.82 1.26 1.12 1.22 1.05 1.22 1.56

0.28 0.28 0.28 0.19 0.30 0.21 0.18 0.21 0.14 0.22 0.23

4.52 5.49 6.17 7.25 7.79 8.36 8.73 9.18 9.76 10.10

4.70 5.67 6.36 7.42 7.96 8.52 8.89 9.33 9.90 10.26

4.81 5.74 6.43 7.45 7.98 8.51 8.86 9.31 9.87 10.23

4.90 5.93 6.65 7.64 8.17 8.70 9.07 9.53 10.07 10.45

3.44 3.23 3.11 2.15 1.96 1.64 1.58 1.57 1.28 1.40 2.13

0.38 0.45 0.48 0.39 0.38 0.34 0.34 0.35 0.31 0.35 0.38

N-glycan nomenclature was adapted from ref 18, and the major IgG glycan structures are highlighted in bold.

Figure 2. Arrhenius plots of logarithmic electrophoretic mobility vs reciprocal absolute temperature for maltooligosaccharides of DP 7-12 (panel A), and the four major IgG glycans (peaks 4, 6, 7, 9, panel B). Capillary electrophoresis separation was accomplished in 25 mM lithium acetate (pH 4.75) background electrolyte with no additives. Solid lines represent the linear least-squares fit of the data (r2= 0.99, each).

Activation Energy Associated with the Electromigration of Linear and Branched Oligosaccharides. Temperature dependent electrophoretic mobilities of linear and branched oligosaccharides were first investigated in a simple background electrolyte of 25 mM lithium acetate (pH 4.75) in the range of 20−50 °C. Figure 1 depicts the resulting capillary electrophoresis traces for the two sample types (maltooligosaccharides, upper trace; IgG glycans, lower trace) at the separation temperatures of 20 °C (panel A) and 50 °C (panel B). While the migration times of both structural groups

significantly decreased with the increase of the temperature, the relative migration time differences between all the branched (lower trace peaks 1−10, representing sialylated, neutral, and core fucosylated biantennary IgG glycans) and linear structures (peaks DP1−DP15, upper trace) were readily apparent. For example, peak 4 of the IgG glycans (FA2) migrated close to the maltooctaose (DP 8) at 20 °C but migrated slightly slower than the maltoheptaose (DP 7) at 50 °C. For easier conception of this phenomenon, the corresponding glucose unit (GU) values were calculated19 and listed in the 11632


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increment for the maltooligosaccharides was 0.4 J/mol per A3, in contrast to the biantennary branched IgG glycans that increased by almost 4 times as much, 1.5 J/mol per A3. We consider these differences as one of the main reasons behind the GU value shift with increasing separation temperature during capillary electrophoresis analysis of these structurally different oligosaccharides. A similar secondary structure change-induced difference in the activation energy values was reported earlier by Dovichi and co-workers for DNA sequencing fragments,22 although in the case of nucleic acids the change was reportedly due to segment differences. Effect of Background Electrolyte Additives. Separation of APTS labeled linear maltooligosaccharides and branched IgG glycans was also evaluated between 20 and 50 °C in 10% ethylene glycol (EG), 2% linear polyacrylamide (LPA, MW 10 kDa), and 0.4% poly(ethylene oxide) (PEO, MW 300 kDa) additive containing 25 mM lithium acetate (pH 4.75) background electrolytes, respectively. Please note that the concentrations of both linear polymer additives (LPA and PEO) were just above their entanglement threshold, measured as reported earlier,23 i.e., should be considered as polymer networks. The viscosity values of the additive containing buffers in the temperature range of the study were 10% EG at 20 and 50 °C = 1.28 mPa s and 0.74 mPa s; 2% LPA at 20 and 50 °C = 1.30 mPa s and 0.82 mPa s; 0.4% PEO at 20 and 50 °C = 2.59 mPa s and 1.32 mPa s, respectively. Similar to as reported above, faster separations were observed in all three instances with increasing temperature but with varied temperature-dependent differential migration between the linear and branched structures as shown in the respective panels in Table 1. While in the additive-free background electrolyte the GU values of the branched glycans changed an average of −0.89 GU in the temperature range investigated, by the addition of 10% ethylene glycol or 2% LPA or 0.4% PEO, the corresponding average GU values shifts were −0.39, + 0.23, and +0.38 units, respectively. Thus, with background electrolytes containing no or only monomeric (ethylene glycol) additive, the GU values of the branched IgG glycans decreased with increasing temperature. On the other hand, when polymeric additives were used in the background electrolyte the GU values of the branched glycans increased with increasing temperature, probably due to possible solute− network interactions or deformation effects in addition to the influence of viscosity. The Arrhenius functions were plotted for all three additive types and the activation energy values associated with the electromigration of the maltooligomers and IgG glycans were calculated from the slopes of the plots. Then the activation energy vs hydrodynamic volume diagrams were plotted (not shown) and the Ea changes were derived for the linear and branched glycans as 0.3 J/mol and 0.9 J/mol per A3, 0.4 J/mol and 1 J/mol per A3 as well as 0.06 J/mol and 0.3 J/mol per A3, respectively, for the ethylene glycol, LPA, and PEO containing separation media. In the instance of the ethylene glycol (monomeric) additive, the activation energy requirement was presumable associated with the passage of the increasing size solute molecules through the higher viscosity background electrolyte. In the presence of the polymer network containing buffer systems, in addition to the viscosity component, based on the obtained activation energy values, other (e.g., physical) interactions between the solute molecules and the polymer network and/or deformation effects should also be considered.

upper left panel of Table 1 (column, 25 mM Li-acetate). In the temperature range of the study, the average glucose unit value shift of the branched glycans was −0.89 GU, i.e., at higher temperatures the complex biantennary structures migrated that much faster than their corresponding maltooligosaccharide counterparts. As a first approximation, the activation energy requirement for the electromigration process of these structurally different sample types was considered to play a crucial role. Therefore, on the basis of eq 5, the Arrhenius diagrams were plotted for the relevant DP range of the maltooligosaccharides (DP 7-12) and the four major biantennary IgG glycans (Peaks 4, 6, 7, 9 of Figure 1) shown in Figure 2, panels A and B, respectively. Maltooligosaccharides (α1-4 linked glucose units) form a full helical structure above DP 7,19 so their structure was considered as linear-helical at the range of this comparative study with the IgG glycans. To shed light on the possible cause of temperature mediated electromigration differences between the linear maltooligosaccharides and branched IgG glycans, the activation energy values were derived from the slopes of the Arrhenius plots in Figure 2 and plotted against the hydrodynamic volumes of the solute molecules shown in Figure 3. In this study we did not consider

Figure 3. Activation energy plots as a function of the hydrodynamic volume of the linear-helical maltooligosaccharides DP 7-12 (●) and the major branched IgG glycans (peaks 4, 6, 7, and 9 in Figure 2) (▲). Solid lines represent the linear least-squares fit of the data (ladder r2 = 0.93; IgG r2 = 0.99). Insets: Molecular structures of the linear maltoundecamer (DP 11, left) and branched FA2G2 IgG glycan (right) show the charge distribution on the surface of the molecular cavity calculated by COSMO.

using the simple degree of polymerization (DP) value to define the size of the branched oligosaccharides as that number would not represent their shape; therefore, the hydrodynamic volumes were calculated. Molecular mechanics optimized structures were taken from the Glycoscience.de database.20 Subsequent single point calculations were carried out at the Density Functional Theory (DFT)/Conductor-like Screening Model (COSMO) level of theory in order to get the realistic molecular volume.21 The structures based on the calculated volumes of the embedding cavity according to the continuum solvation model are shown in the insets of Figure 3 for the maltoundecaose and the FA2G2 IgG glycan. Figure 3 depicts the changes in activation energy requirement of the analyte molecules to migrate through the separation medium for both solute types. The activation energy 11633


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(21) Klamt, A. From Quantum Chemistry to Fluid Phase Thermodynamics and Drug Design; Elsevier: Amsterdam, The Netherlands, 2005; pp ix−xi. (22) Lu, H.; Arriaga, E.; Da, Y. C.; Figeys, D.; Dovichi, N. J. J. Chromatogr., A 1994, 680, 503−510. (23) Guttman, A.; Cooke, N.; Starr, C. M. Electrophoresis 1994, 15, 1518−1522.

CONCLUSIONS To elucidate the interesting phenomenon of temperature dependent GU value shifts of branched glycans in capillary electrophoresis without and with the presence of monomeric or polymeric background electrolyte modifiers, the activation energy changes associated with the electromigration of these different oligosaccharide structures were investigated. On the basis of our results, we suggest that the GU value shifts were apparently caused by the temperature-dependent activation energy requirement for the linear and branched sugar structures to migrate through the separation media investigated. This emphasizes the high importance of tight temperature control during glycan analysis by capillary electrophoresis if GU values form existing databases are used for structural elucidation.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +36 (88) 624-063. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the MTA-PE Translation Glycomics (Grant No. 97101) and the NKFIH (Grant K 116263) grants of the Hungarian Government. The authors gratefully acknowledge Professor Pierre Gareil for his stimulating discussions.

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REFERENCES

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Effect of Separation Temperature on Structure Specific Glycan

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