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Microscale Exoglycosidase Processing and Lectin Capture of Glycans with Phospholipid Assisted Capillary Electrophoresis Separations S. A. Archer-Hartmann,† L. M. Sargent,‡ D. T. Lowry,‡ and L. A. Holland*,† † ‡
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, West Virginia 26505, United States
bS Supporting Information ABSTRACT: Capillary electrophoresis separations of glycans labeled with 1-aminopyrene-3,6,8-trisulfonic acid were achieved with separation efficiencies ranging from 480 000 to 640 000 theoretical plates in a 60.2 cm, 25 μm inner diameter fused silica capillary. Under these separation conditions, the coefficient of variation in peak area is 10%, and if labeling efficiency is estimated at 100%, the limit of detection is 15 fM. The capillary electrophoresis method incorporated phospholipid additives to enhance the separation of glycans with slight differences in hydrodynamic volume. In addition, the phospholipid additives supported the integration of the lectin concanavalin A as well as the enzymes R1-2,3 mannosidase or β1-4 galactosidase to provide structural and compositional information about the glycans subject to separation. The use of in-capillary cleavage of terminal glycan residues with exoglycosidases offers a number of advantages over benchtop enzymatic sequencing, including reduced consumption of analyte, as well as enzyme. These methods were used to evaluate glycans derived from the glycoproteins R1-acid glycoprotein, fetuin, and ribonuclease B, as well as from glycoproteins collected from MCF7 cells.
A
nalysis of glycan composition is essential to therapeutics, and glycans hold potential as effective biomarkers for a variety of diseases. For example, a change in glycosylation pattern is a hallmark of cancer, but the change in glycosylation can be strikingly different for different types of cancer and different stages of the cancer. Cancerous tissues can display a variety of changes such as β1-6 mannose branching, fucosylation, increased truncation with sialic acid capping, or sialylated Lewis type antigens composed of fucose, galactose, and N-acetylglucosamine.1 3 While there is a fundamental need to profile glycans in cancerous tissues, glycan analysis is not straightforward. This is because of the complexity of the glycan structure, which is defined by the anomeric form of the saccharide monomers, variation in the type of monomeric saccharide unit, the position of the linkage between adjacent saccharide monomers, and chain branching. Mass spectrometry is one tool that can be used to study glycoconjugates4 or glycan sequence, but the challenge of linkage analysis, isomerization, and interpretation must be overcome with sophisticated analyses.5 7 Sequence identification with electrospray ionization is complicated by the appearance of different charge states, ion suppression, adduct formation, and fragmentation. Matrix assisted laser desorption ionization typically requires derivatization to prohibit extensive fragmentation during ionization, and quantitative analyses require isotope labeling. While matrix assisted laser desorption ionization must be coupled off-line with analytical separation methods, often via fraction collection, electrospray ionization methods can be r 2011 American Chemical Society
coupled online to chemical separations. Isobaric glycans are common and cannot be resolved except with complex derivatization or tandem mass spectrometry.8 Enzymatic analyses with exoglycosidases may distinguish the structures of isobaric glycans. Mass spectrometry sequencing by sequential analyses following enzyme treatment can be used with electrospray ionization coupled with an ion trap9,10 or matrix assisted laser desorption ionization coupled with time-of-flight.11 14 These methods may be modified to eliminate the need for desalting following enzymatic processing.12,15 Enzymatic sequencing can be performed on the target used for matrix assisted laser desorption ionization but requires 0.5 μL of each enzyme per spot.16 Although benchtop enzyme sequencing can be used to determine glycan sequence,9 the reactions are daunting because scaling up the labeling chemistry is expensive, the duration of the cleavage reaction can be lengthy, and the purification procedure is laborious.17 Ultimately, strategies that separate the sample components into singlet peaks, reduce the total amount of sample handling, and require small sample volume are critical for such studies. When enzymatic analyses are fully integrated into the separation device, loss in sample transfer is minimized, as is the time required for enzymatic reaction. The sample to be analyzed is generally volume-limited with low endogenous Received: December 29, 2010 Accepted: February 23, 2011 Published: March 15, 2011 2740
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Analytical Chemistry concentrations of glycans, in a complex biological matrix, and composed of a mixture of oligosaccharides with only subtle differences in sequence as well as composition. These analytical challenges present a critical barrier to effectively identify and quantify changes in the glycan content of relevant biological samples. The first step to elucidating glycan composition in biological samples is a rapid microscale separation method compatible with biological methods of selection. Capillary electrophoresis is a rapid and efficient method of separation for carbohydrate analyses,18 20 that has been coupled with benchtop exoglycosidase sequencing to elucidate glycan composition.21 23 As a microscale technique, it is particularly suited for biological samples because it requires nanoliter to picoliter injections. However, sample volumes that are more practical to handle (20 30 μL) are generally used. A 20 μL sample can be injected repeatedly with a capillary electrophoresis system, without significant sample loss. The fill volume of a 60 cm long, 25 μm inner diameter capillary is only 0.3 μL, and each anodic and cathodic reservoir vial generally contains a running buffer volume less than 1.5 mL. Polysaccharides, including glycans, are frequently labeled with a charged fluorescent dye to impart electrophoretic mobility to neutral saccharides and to realize the low limits of detection inherent with laser induced fluorescence.18,19,24 28 One example, 1-aminopyrene-3,6,8-trisulfonic acid (APTS), is commonly reported in the literature.29 With literature protocol, the labeling efficiencies range from 80 to 95%30 32 for linear or branched31,32 oligosaccharides and the labeling reaction is reproducible enough for quantitation. Electrophoretic glycan separations in capillary are typically performed under reversed polarity in conjunction with surface passivation to suppress electroosmotic flow. Different additives have been incorporated in capillary electrophoresis to enhance the separation; however, most separations are limited to capillaries larger or equivalent to 50 μm inner diameter. This is because covalently modified capillaries of smaller inner diameters are not commercially available and are difficult to fabricate in-house. Recently, semipermanent phospholipid coatings have been described that passivate the capillary surface and suppress bulk electroosmotic flow. These phospholipid coatings are simple to apply and are easily adapted to capillary of a variety of inner diameters.33 36 Previously, the potential of phospholipid additives for capillary electrophoresis was demonstrated and the effects of temperature, phospholipid content, and composition on separation performance were investigated.37 In this article, phospholipids were used as an additive to enhance glycan separations and to incorporate lectins or enzymes to provide structure and sequence information about glycans using a 25 μm inner diameter capillary. Phospholipid additives are compatible with proteins and are easily integrated with enzymes and lectins to provide insight into glycan composition. The enzymes β1-4 galactosidase and R1-2,3 mannosidase and the lectin concanavalin A were incorporated in efficient separations of glycan standards. The strategy was applied to MCF7 cells, which are immortalized cells derived from human breast tumor. These cells are known to display aberrant glycan composition and serve as a test bed to demonstrate the potential of phospholipid additives to support compositional analyses and efficient glycan separations.
’ MATERIALS AND METHODS Chemicals. Maltooligosaccharide standards, concanavalin A from Canavalia ensiformis, 3-(N-morpholino)-propanesulfonic acid (MOPS), asialofetuin, sodium chloride, sodium hydroxide,
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tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), Triton, and MgCl2 were purchased from Sigma-Aldrich (St. Louis, MO). Methanol, CaCl2, and R1-acid glycoprotein (AGP) were purchased from EMD Biosciences (La Jolla, CA). Ethanol was purchased from AAPER Alcohol (Shelbyville, KY). The lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were purchased from Avanti Polar Lipids (Alabaster, AL). APTS was purchased from Biotium (Hayward, CA). Sodium cyanoborohydride (NaCNBH3) was purchased from Strem Chemicals (Newburyport, MA). Triethylamine, acetonitrile, and citric acid were purchased from Fisher Scientific (Pittsburgh, PA). Fetal bovine serum was obtained from Thermo Scientific (Rockford, IL). Ribonuclease B (RNase B), β1-4 galactosidase, R1-2,3 mannosidase, a peptide N-glycosidase (PNGase F) kit, and a neuraminidase kit were purchased from New England Biolabs (Ipswich, MA). The PNGase F kit was supplied with a G7 10X reaction solution composed of 0.5 M sodium phosphate buffered at pH 7.5, a glycoprotein denaturing solution containing 5% sodium dodecyl sulfate and 0.4 M dithiothreitol, and a 10% solution of the nonionic surfactant NP-40. The neuraminidase kit was supplied with a G1 10X reaction solution composed of 0.5 M sodium citrate buffered at pH 6. Deionized water was obtained from an Elga Purelab ultra water system (Lowell, MA). Preparation of Phospholipid Additive. Phospholipids were prepared as described previously, aliquoted, and stored at 20 °C.38 Briefly, the appropriate mass of DMPC and DHPC was combined in a centrifuge tube. The dry phospholipid was hydrated with 100 mM aqueous MOPS buffered at pH = 7. The preparation was thoroughly mixed using a vortex mixer. After mixing, the preparation underwent a minimum of three freeze thaw cycles and was centrifuged for 10 min at 10 000 rpm. Prior to use, samples were thawed and vacuum degassed for 1 min. Preparations used for coating the separation capillary consisted of 5% phospholipids by mass at a molar ratio of DMPC to DHPC of q = 0.5. Preparations used as an additive for electrophoretic separation consisted of 10% phospholipids by mass at a molar ratio of DMPC to DHPC of q = 2.5. All phospholipid preparations were made in 100 mM MOPS buffered at pH 7.0. Assuming a specific volume of phospholipid is ∼1 mL/g,39 the total phospholipid concentration of the q = 0.5, 5% and q = 2.5, 10% preparations was 90 mM (30 mM DMPC, 60 mM DHPC) and 148 mM (106 mM DMPC, 42 mM DHPC), respectively. Preparation and Derivatization of Standards. Linear maltooligosaccharides including maltotetraose (G4), maltopentaose (G5), malothexaose (G6), and maltoheptaose (G7) were obtained from a commercial source. However, branched glycans used as standards (see Figure 1 for structures) were derived from the glycoproteins AGP, fetuin, and RNase B. AGP and fetuin yield sialylated hybrid asparagine-linked (N-linked) glycans that are biantennary (AGP, fetuin), triantennary (AGP and fetuin), or tetraantennary (AGP). These branched hybrid bi-, tri-, and tetraantennary glycans derived are abbreviated as AI, AII, AIII, AIV, and AV. RNase B yields N-linked high mannose glycans. These are mannose 5 (Man5), mannose 6 (Man6), mannose 7 (Man7), mannose 8 (Man8), and mannose 9 (Man9). Branched glycan standards were collected from glycoproteins using a PNGase F kit according to the manufacturer’s instruction, with the exception that the cleavage reaction was incubated overnight at 37 °C. A 3 volume of ice-cold ethanol was added to the cleavage reaction and centrifuged for 10 min at 10 000 rpm at 2741
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Figure 1. Electropherogram of standard glycans labeled with APTS. The structure for branched glycans associated with each peak is included in the figure. Mannose 5, mannose 6, mannose 7, mannose 8, and mannose 9 are abbreviated Man5, Man6, Man7, Man8, and Man9, respectively. Branched bi-, tri-, and tetraantennary glycans derived from AGP are abbreviated as AI, AII, AIII, AIV, and AV. Peaks labeled G4, G5, G6, and G7 are maltotetraose, maltopentaose, malothexaose, and maltoheptaose, respectively. Separation conditions are described in the text.
4 °C to precipitate the protein. The fraction containing the cleaved glycan was removed and dried with gas (nitrogen or argon). Once dried, the glycans were reconstituted in a 1 μL volume of a 0.2 M solution of APTS dissolved in 1.2 M citric acid. This was combined with a 1 μL volume of a 1 M solution of NaCNBH3 dissolved in deionized water. The mixture was thoroughly mixed with a vortex mixer and incubated at 55 °C for 2 h. The reaction was stopped with the addition of 100 μL of deionized water. The solution was adjusted to a pH of 6 using sodium hydroxide, and 100 units (2 μL) of neuraminidase was added. The solution was incubated overnight at 37 °C. The next day, the solution was filtered through a 10 kDa molecular weight cut off filter (Sartorius, Stonehouse, UK) to remove the neuraminidase. Excess APTS was removed from the filtered solution using either a 1 kDa molecular weight cut off filter (Pall, Port Washington, NY) or by a Discovery DPA-6S solid phase extraction cartridge (50 mg packing material, Supelco, Bellefonte, PA). Solid phase extraction was carried out on the basis of the modification of literature procedures.40,41 Briefly, the DPA-6S SPE tube was flushed with 95:5 (v/v) acetonitrile/deionized water. The glycan solution was diluted to ensure a final composition of 95% acetonitrile prior to the purification process. Once loaded in the extraction cartridge, the APTS was eluted using a solution composed of 95% acetonitrile and 5% deionized water that also contained 50 mM triethylamine. The retained glycans were eluted from the cartridge using a solution of 50 mM triethylamine in deionized water. Following purification, the glycan solution was dried using a Savant SpeedVac concentrator (ThermoScientific, Waltham, MA), reconstituted in 100 μL deionized water, and stored at 20 °C. Prior to injection, sample was diluted at least 8-fold in MOPS and stored at ambient temperature or 4 °C. Culture of Cells and Protein Isolation. MCF7 cells (ATCC, Manassas, VA) were cultured with Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 10%
fetal bovine serum. Total cellular protein was isolated from approximately 10 106 MCF7 cells in a T75 flask. Two mg/mL of total protein was isolated when the cells were approximately 70% confluent. The protein was extracted in 50 mM Tris HCl, 1% Triton, 1.5 mM MgCl2, 150 mM NaCl, and protease inhibitors following procedures outlined previously.42 An 83 μg sample of the isolated protein fraction taken from the MCF7 cells was prepared using the procedure outlined to label glycan standards. Following labeling, excess APTS and APTS labeled small molecules (e.g., monosaccharides) were removed using 1 kDa molecular weight cutoff filters. Capillary Electrophoresis. Analyses were performed using a Beckman Coulter P/ACE MDQ (Beckman Coulter, Fullerton, CA) configured by the manufacturer with laser induced fluorescence detection (3 mW air cooled argon ion, λex = 488 nm, λem = 520 nm). A 25 μm internal diameter, 360 outer diameter fused silica capillary (Polymicro Technologies, Phoenix, AZ) was used for separation. Each day, capillaries were prepared by flushing with the following sequence: 1 M NaOH for 30 min at 170 kPa (25 psi), deionized water for 15 min at 170 kPa (25 psi), 15 min with methanol at 170 kPa (25 psi), and deionized water for 15 min at 170 kPa (25 psi). The capillary was then coated with q = 0.5, 5% phospholipid containing 1.25 mM calcium for 20 min at 170 kPa (25 psi), followed by a 2 min, 170 kPa (25 psi) rinse with MOPS. The phospholipid preparations have low viscosity below the gel phase transition temperature and were easily introduced in the capillary. Prior to each run, the capillary was held at 19 °C and flushed as follows: 3 min of q = 0.5, 5% coating phospholipid at 170 kPa (25 psi), 2 min of MOPS at 170 kPa (25 psi), 3 min of q = 2.5, 10% phospholipid. Experiments that required the introduction of enzyme or lectin into the capillary used the same flush sequence except a 3 min, 170 kPa (25 psi) MOPS flush was additionally completed in the reverse direction to push out any remaining 2742
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Analytical Chemistry protein. Ambient thermal control of the room and instrument was maintained using a portable air conditioner as was described previously.37 After the capillary was filled with q = 2.5, 10% phospholipid at 19 °C, the temperature of the separation cartridge was increased to 25 °C for the injection and separation. Injection was completed in three steps, as was previously reported.37 A preplug of MOPS was injected into the phospholipid-filled capillary, unless otherwise noted at 6.9 kPa (1.0 psi) for 7 s, followed by the electrokinetic injection. Following injection, a postplug of MOPS was injected for 3 kPa (0.5 psi) for 5 s. Unless otherwise noted, separation was carried out at 400 V/cm (reversed polarity). Data collection and analysis were performed using 32 Karat Software version 5.0 (Beckman Coulter). Theoretical plates were calculated using 32 Karat Software using the “USP plates” criterion.
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Table 1. Effect of Injection Size on Precision and Plate Counta injection condition
precision: areab
0.1%
10%
537 000
1%
6%
485 000
10 kV 3 s 4 kV 5 s
theoretical plates
a
Mannose 6 (from RNase B), n = 5. The effective length (Leff) was 50.0 cm. The total length (Lt) was 60.2 cm. The applied voltage was 400 V/cm. The anodic/cathodic reservoir contained 100 mM MOPS buffered at pH = 7, and the capillary was filled with 10% q = 2.5 phospholipid as described in the text. b Reported as relative standard deviation.
Table 2. Effect of Length on Separation Efficiencya
’ RESULTS AND DISCUSSION Separation Conditions for Glycan Standards. Phospholipid
assisted separations were previously characterized, and it was determined that a separation additive of 10% phospholipid solution with [DMPC]/[DHPC] = 2.5 yielded the best separation performance (240 000 theoretical plates for AII glycan).37 The separation, based on hydrodynamic volume, was accomplished using a 50 μm inner diameter capillary37 but should also be compatible with a 25 μm inner diameter capillary. Advantages to the use of a 25 μm inner diameter capillary include reduced consumption of phospholipid additives and reduced separation current. For the integration of exoglycosidase with in-capillary cleavage, a chief benefit to using the 25 μm inner diameter capillary is the reduction of the consumption of enzyme. Previously, the success of glycan separations performed with a 50 μm inner diameter capillary depended on the application of hydrodynamically introduced injection pre- and postplugs of 34 kPa (5.0 psi), 7 s and 3 kPa (0.5 psi), 5 s, respectively.37 Without these bracketed injections, the sample cannot be predictably injected into the capillary and the analyte peaks are substantially broadened. This injection phenomenon has been noted in other reports in which the preplug creates an ion depleted zone that serves to improve sample introduction,43 while incorporation of a postinjection aqueous plug reduces peak tailing resulting from carryover.44 To maintain efficient separations and reproducible injections, these parameters were optimized for a 25 μm inner diameter separation capillary. Two different preplug volumes were implemented by applying a pressure of 69 kPa (10. psi) or 3 kPa (0.4 psi) for 7 s to inject mannose 6 electrokinetically (10 kV 3 s) into a 30.2 cm long capillary. No statistical difference in efficiency or peak area was obtained until the applied pressure and duration of the preplug were lower than 3 kPa (0.4 psi) and 7 s (see Table S-1 in the Supporting Information); therefore, injections were accomplished using a 6.9 kPa (1.0 psi) 7 s MOPS preplug. Unlike the results obtained using a 50 μm inner diameter capillary, the use of a postinjection plug did not affect separation efficiency in 25 μm inner diameter capillary. However, the postinjection plug was used for separations in the smaller inner diameter capillary, as it did not increase the analysis time. Electrokinetic Injection. The time of the electrokinetic injection affects separation efficiency as smaller injection bands reduce the peak broadening associated with sample introduction. However, the variance associated with fast injections as well as the instrumental ramp time associated with the application of voltage affects the amount of sample injected electrokinetically
precision: timeb
Leffb
timec (RSD)d
plates (RSD)d
10.2
3.11 (1%)
32 000 (3%)
320 000
20.0
6.05 (0.2%)
133 000 (2%)
665 000
30.0
8.88 (0.7%)
234 000 (3%)
780 000
40.0 50.0
12.14 (0.4%) 15.06 (0.1%)
378 000 (1%) 530 000 (2%)
945 000 1 060 000
plates per meter
a
Mannose 6 (from RNase B), n = 5; sample injected for 10 kV 3 sec; other conditions as described in Table 1. b In cm. c In minutes. d Relative standard deviation.
into the capillary and, hence, the reproducibility of peak area. This effect is demonstrated with glycans derived from RNase B injected for either 10 kV 3 s or 4 kV 5 s (see Table 1). On the basis of these results, electrokinetic injection conditions that yielded the highest separation efficiency (10 kV for 3 s) were used. When the quantitation of peak area is important, coefficients of variation of less than 6% can be achieved with a 4 kV 5 s injection (see Table 1); however, the theoretical plate count decreases. The limit of detection of the method was estimated for a 10 kV 3 s injection using a linear maltooligosaccharide (maltopentaose). The oligosaccharide was labeled with APTS as the limiting reagent. If the labeling efficiency was 100%, then a 300 femtomolar sample of maltopentaose electrokinetically injected into the separation capillary (10 kV 3 s) yielded a S/N of 60 and a limit of detection of 15 femtomolar. Effect of Capillary Length. Analyte resolution improves with increasing capillary length45 or increasing ratio of effective tube length to total length.46 The effect of capillary length was characterized using branched glycans derived from RNase B (Table 2) and linear maltooligosaccharides (see Table S-2 in the Supporting Information). The total capillary length varied from 30.2 to 60.2 cm, while the effective capillary length varied from 10.2 to 50.0 cm. As the effective separation length increased, the migration time also increased. A theoretical plate count of 530 000 was obtained using a 60.2 cm long capillary and an effective length of 50.0 cm. When the data are normalized to plates/meter to account for length, the separation efficiency also increased with capillary length. Separations were performed on a capillary with a total length of 60.2 cm, but sample was introduced at the end of the capillary that produced an effective length of 10.2 or 50.0 cm. When the total length is constant but the effective length changed, higher theoretical plate count was obtained with higher effective separation length. There was no statistical difference in the separation efficiency obtained with branched or linear glycans (see Supporting Information Table S-2). On the basis of these data, glycan separations were accomplished 2743
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Figure 2. Effective integration of enzymatic cleavage and phospholipidenhanced separations of APTS labeled glycans. In both traces, asialoglycan isomers AII and FII derived from fetuin are baseline resolved. The separation in the solid trace was obtained using a separation capillary filled with phospholipid additive and a small buffer plug loaded with the enzyme β1-4 galactosidase. The separation in the dashed trace was obtained using a separation capillary filled with phospholipid additive and a small buffer plug that serves as a blank. Separation conditions are described in the text. Both separations were accomplished using a 15 min in-capillary incubation prior to separation.
using a 60.2 cm capillary and an effective separation length of 50.0 cm. Effect of Applied Electric Field. In capillary electrophoresis, increased separation efficiency is obtained when the applied voltage is as high as possible yet does not induce excessive Joule heating. The viscosity of the phospholipid media used in these glycan separations was temperature dependent and could potentially also be affected by Joule heating. Therefore, the absence of significant Joule heating was verified. Because the maximum applied voltage for the commercial instrument used for this work was 30 kV, a shorter capillary (40.0 cm) was used so that the full range of applied electric field could be interrogated to determine the effects of higher field strength. Figure S-1 in the Supporting Information is a plot of plate height versus electric field strength. The lowest plate heights were obtained from 350 to 450 V/cm. This is consistent with the separation suffering from longitudinal band broadening at the field strengths below 350 V/cm and from Joule heating at field strengths above 450 V/cm. Subsequently, glycan separations were performed using an effective field strength of 400 V/cm. Separations accomplished using a 60.2 cm capillary required 24 kV applied voltage. Separation and Identification of Glycan Standards. The optimized injection and separation conditions were used to obtain the separation of glycan standards shown in Figure 1. Analyte peaks are labeled in the electropherogram. Additional peaks most likely emanate from impurities extracted from plastic used during the labeling process.22 The theoretical plate count of
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the analyte peaks ranged from 480 000 for AV to 639 000 for AIII and was a considerable improvement over separation efficiencies attained by capillary electrophoresis separations achieved in fused silica capillaries and reasonable improvement over the most efficient separations reported in microchips to date.37,47 The phospholipid media does not denature proteins and can be used to integrate lectins or enzymes for additional selection and to facilitate peak identification. To verify this compatibility in a 25 μm inner diameter capillary, the separation capillary was loaded with phospholipid and then with a small plug of the enzyme β1-4 galactosidase reconstituted in 50 mM citrate buffered at pH 6. Following injection, the sample was moved into the enzyme plug by briefly applying electric field (10 kV 30 s). The glycan was incubated in the capillary for 15 min and then separated. Blank enzyme reactions were performed to compare peak areas. This was originally accomplished using either heat-denatured enzyme or citrate buffer loaded in place of the enzyme. The performance of both methods was equivalent, so in most cases, a buffer cartridge was used rather than deactivated enzyme. The overlaid traces in Figure 2 were obtained in the presence (solid line) and absence (dashed line) of enzyme, respectively. In spite of the diffusion inherent with a 15 min in-capillary incubation, the separation efficiency was high in both traces. For example, the FII glycan subjected to a 15 min incubation blank had a theoretical plate count of 310 000, while in the presence of enzyme the plate count decreased to 190 000. FII and AII glycan have the same monomeric composition and structure. However, the terminal galactose in the middle branch (labeled with an asterisk in Figure 2) of the AII glycan is β1-4 linked to N-acetylglucosamine, while in FII glycan it is β1-3 linked to N-acetylglucosamine. In both traces, the resolution, defined as the difference in the migration time divided by the average width of the peak base, of this isomer pair is 1.0. Enzymatic cleavage of at least one of the three terminal β1-4 galactose residues of the AII glycan occurs rapidly during the 15 min incubation, and the peak area of AII in the enzyme-treated and blank sample decreases from 76 000 to 8400. For the FII glycan, which possesses a single β1-3 linked galactose, following incubation with enzyme, the peak area decreases from 42 000 to 11 000. In addition, new peaks appeared at 17.21 and 17.40 min that increased from 8200 to 41 000 and from 4200 to 14 000, respectively. The progress of the enzymatic cleavage was dependent upon the incubation time. This is shown in the Supporting Information in Figure S-2, which contains overlaid traces at 0, 5, 10, and 15 min of incubation. The corresponding areas obtained in each trace are summarized in Table S-3 also in the Supporting Information. The enzyme plugs may be replaced with lectin, such as concanavalin A which binds with high affinity to high mannose glycans and glycans with mannose branching. A small plug of concanavalin A reconstituted in MOPS was injected into the separation capillary after it was loaded with phospholipid media. A mix of glycan standards, which included high mannose glycans (Man5 9) was subjected to concanavalin A capture or a blank capture in Figure 3. Unlike enzyme treatment, lectin capture does not require incubation. The sample subjected to concanavalin A capture (upper trace) was devoid of the mannose glycans that appear in the blank (lower trace). Identification of Glycans in MCF7 Cells. On the basis of the data obtained using glycan standards, the method was applied to glycans derived from MCF7 cells. MCF7 cells are commonly used as a model cell line to characterize advances in instrumentation and 2744
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Figure 3. Separation of APTS labeled glycan standards including the linear maltooligosaccharides and branched glycans taken from the glycoproteins RNase B and AGP. The bottom trace displays all resolved glycans in the separation. The upper trace shows the glycan standard separation in the capillary containing a small plug of the lectin concanavalin A. Separation conditions are described in the text.
Figure 4. Electropherogram of the labeled glycan fraction derived from MCF7 cells. Glycan composition is interrogated using the enzymes β1-4 galactosidase or R1-2,3 mannosidase or the lectin concanavalin A. The response of analyte peaks to enzymes or lectin is labeled in the figure. Separation conditions are described in the text.
methodology in the field of proteomics. The glycan composition has been investigated in this cell line.48 Notably, much of the
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glycan content derived from MCF7 cells is high mannose glycan. No attempt was made to remove saccharides present in the sample prior to sample reaction. The sample was desialylated prior to labeling with APTS. To decrease the amount of excess labeling reagent, the sample was purified with a 1 kDa molecular weight cutoff filter. Treatment with the polymeric DPA-6S extraction cartridges was not used, as it resulted in loss of the low abundance glycan peaks. Following sample preparation and purification, the MCF7 sample was separated using phospholipid-assisted capillary electrophoresis. The region of the electropherogram rich in high mannose and hybrid glycans is shown in Figure 4 and in supplemental Figure S-3 in the Supporting Information. The sample was spiked with glycan standards derived from RNase B and AGP. The peaks that were identified with these standards are labeled in Figure 4. Glycan composition was investigated with enzymes and the lectin concanavalin A. The coefficient of variation of peak area was less than 10%. Therefore, the criterion for response to enzyme or lectin is a decrease in peak area greater than or equal to 15%. Glycans that possessed a terminal β1-4 galactose residue were identified with a 10 min in-capillary incubation of the MCF7 glycan sample with β1-4 galactosidase. Each peak that decreased in area by more than 15% following enzyme treatment is labeled in the figure. Incapillary enzyme incubation was repeated with R1-2,3 mannosidase. Glycans that possessed a terminal mannose residue were identified following a 15 min incubation of the MCF7 glycan sample. Analyte peaks that decreased in area by more than 15% following enzyme treatment are labeled. The mannose composition of the glycans derived from MCF7 cells was further verified using an off-capillary incubation of the glycans derived from MCF7 cells with concanavalin A. The analyte peaks that displayed a decrease in area greater than the coefficient of variation are labeled in the figure. The analyte peak identified as Man5 failed to respond to concanavalin A. This may be due to the low abundance of the Man5 and competing affinity of higher abundance mannose rich and mannose branched glycans, although others have noted that lectins display unusual interactions that do not obey predicted epitope binding.49 The lectin concanavalin A, as well as the enzymes R1-2,3 mannosidase and β1-4 galactosidase, provided intriguing information about the glycans derived from the soluble glycoproteins in the MCF7 cell line. The glycans associated with branched mannose structure similar to those found in RNase B appeared in the same region of the electropherogram and responded to concanavalin A, which selects for high mannose and mannose branched glycans. In-capillary cleavage of the sample with the exoglycosidase β1-4 galactosidase was observed for both high and low abundance peaks, while response to in-capillary digestion with R1-2,3 mannosidase was primarily observed for the high abundance glycan peaks. This may reflect differences in glycan composition. A number of glycans in the sample responded to more than one type of protein selection, which provides insight into structure. Examples of combined responses indicate structural features that include branched mannoses, terminal mannose, and/or terminal galactose monomers. High mannose and hybrid branched glycans have been reported in MCF7 cells.48 The capillary electrophoresis method is suitable for highly complex, low abundance biological samples. Ongoing research includes characterization of the maximum rate of incapillary enzymatic cleavage for different enzymes as well as different samples. The complexity of this sample demonstrates 2745
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Analytical Chemistry the benefit of the use of enzymes and lectins in combination with the electrophoresis separation to probe glycan composition.
’ CONCLUSIONS The incorporation of exoglycosidase enzymes and lectins is an essential strategy to identify glycans by composition and structure. Capillary electrophoresis utilizing phospholipid additives provides efficient separations of standard glycans derived from common glycoproteins as well as glycans derived from glycoproteins produced in the MCF7 breast cancer tumor cell line. The highest separation efficiencies were obtained with 25 μm inner diameter capillary with the longest effective separation length. The compatibility of a semipermanent phospholipid surface coating is critical to the use of a separation capillary with an inner diameter of 25 μm, as covalently modified capillaries of this diameter are not easily obtained commercially. The use of a smaller inner diameter capillary reduces the separation current, as well as sample and reagent consumption. As semipermanent surface coatings have recently been demonstrated with 10 μm inner diameter capillaries,50 experiments are currently underway to realize the benefits of incorporating these additives in even smaller diameter capillary. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional results include injection optimization, the effect of separation length on linear oligosaccharides, the effect of electric field on separation efficiency, glycan cleavage at different incubation times, and the electropherogram shown in Figure 4 with the scale reduced to accommodate all analyte peaks. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This material is based upon work supported by the National Science Foundation under Grant No. CHE0749764. This work was supported in part by a grant from the West Virginia Graduate Student Fellowships in Science Technology Engineering and Math (STEM) program to S.A.A.-H. The authors wish to acknowledge the excellent suggestions made by internal reviewers at the National Institute for Occupational Safety and Health. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health. ’ REFERENCES (1) Peracaula, R.; Barrabes, S.; Sarrats, A.; Rudd, P. M.; de Llorens, R. Dis. Markers 2008, 25, 207–218. (2) Fuster, M. M.; Esko, J. D. Nat. Rev. Cancer 2005, 5, 526–542. (3) Dube, D. H.; Bertozzi, C. R. Nat. Rev. Drug Discovery 2005, 4, 477–488. (4) Lazar, I. M.; Lazar, A. C.; Cortes, D. F.; Kabulski, J. L. Electrophoresis 2011, 32, 3–13. (5) Sheng, Q.; Mechref, Y.; Li, Y.; Novotny, M. V.; Tang, H. Rapid Commun. Mass Spectrom. 2008, 22, 3561–3569.
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