A New Monolithic ConA Affinity Column for Purification and Analysis of Glycans, Glycopeptides, and Glycoproteins Srinivasa Rao, Kelly Flook, Andy Woodruff, Yury Agroskin, and Chris Pohl, Dionex Corporation, Sunnyvale, CA

INTRODUCTION Lectin-affinity chromatography has been widely used for the purification Binding Site Binding Site and analysis of oligosaccharides, glycopeptides, and glycoproteins. However, most of the lectin-affinity columns currently available are agarose bead-based spin columns, which have to be operated manually and can only be used for a limited number of purification cycles. With the growing interest in glycoproteomic studies, such as biomarker identification, there is an increasing need for a robust HPLC lectin column. Presented here is the development and applications of a new monolithic Concanavalin A (Con A) affinity column. Concanavalin A is a lectin derived from Canavalia ensiformis (Jack bean) seeds. At neutral and alkaline pH, Con A exists as a tetramer of four identical subunits with a total molecular weight of approximately 104 kDa. Below pH 5.6, Con A dissociates into active dimers of 52 kDa. Con A is one of the most well characterized and widely used lectins. It binds to α-mannose, and to α-glucose with weaker affinity. Divalent metal ions such as calcium (Ca2+) or magnesium (Mg2+) Binding Site Binding Site need to be present to keep Con A active for its binding to carbohydrates. 28188 Figure 1 shows the four monomer units, each of which binds a calcium and a transition metal, typically manganese. The high-capacity ProSwift® Figure 1. Structure of Concanavalin A at 2.4A resolution; Hardmad, K.D.; ConA-1S column can provide fast and efficient purification and analysis for Ainsworth, C.F. Biochemistry, 1972, 11, 4910–4919. various Con A-binding glycoconjugate samples. The HPLC compatibility of this column allows automatic sample injection, high throughput, and excellent reproducibility. Column: ProSwift ConA-1S (5 × 50 mm) Eluent A: 50 mM NaOAc, 0.2 M NaCl, pH 5.3, Column: ProSwift ConA-1S (5 × 50 mm) Flow Rate: 0.4 mL/min with 1 mM CaCl2, 1 mM MgCl2 Eluent B: 100 mM α-Methyl-mannopyranoside Eluents: A. 50 mM NaOAc, 0.2 M NaCl, pH 5.3, in eluent A 1 mM CaCl2, 1 mM MnCl2 Gradient: 100% A from 0–1 min, 1% B from 1.5–6 min, B. 125 mM α-Methyl-mannopyranoside 100% A from 6.5–15 min in eluent A Flow Rate: 1 mL/min C. 200 mM Galactose in eluent A 1 Temperature: 25 ˚C Gradient: Binding - 100% A Injection Vol.: 15 µL Eluting - Step to 100% B or C as Detection: UV at 305 nm indicated by arrows Sample: 25 µg/mL pNP-labeled α,β-glucose, Temperature: 25 °C pNP-labeled α-mannose Sample: 0.5 mg/mL Horseradish peroxidase (HRP) Detection: UV at 403 nm Inj. Volume: 40 µL Peaks: 1. pNP-β-glucose HRP 2. pNP-α-glucose 3. Unidentified impurity 4. pNP-α-mannose

HRP 4 mAU mAU 2 125 mM Man 200 mM Gal

200 mM Gal 3

125 mM Man

012 3 4 5 6 0 5 10 15 Minutes Minutes 27551 27453

Figure 2. Specificity of the ProSwift ConA-1S column. Separation of Figure 3. Specificity of the ProSwift ConA-1S column. Specific elution of three pNP-sugars. horseradish peroxidase. system requirements COLUMN CHEMISTRY AND SPECIFICITY The ProSwift ConA-1S column can be used on any compatible HPLC The ProSwift ConA-1S column is a polymeric monolith prepared by system, which usually consists of a gradient pump; an autosampler; a in-column polymerization, followed by functionalization with Con A. thermal compartment; and a UV, fluorescence, or other type of detector, The monolith is a cylindrical polymer rod containing uninterrupted, depending on the sample type. interconnected, flow-through pores, with surface area greater than nonporous bead-based columns. The structure consists of small COLUMN SPECIFICATIONS pores that contribute surface area, and larger pores that allow reduced backpressure at elevated flow rates. This approach results in short Column Dimension: 5 × 50 mm mass-transfer distances that produce improved efficiency, even at Protein Coated on Monolith: Concanavalin A elevated flow rates. High quality Con A is covalently attached to the Bimodal Monolith Pore Diameter: 3.25 and 0.62 µm monolith column through the amine groups of Con A (see Figure 1). Binding Capacity: ~2 mg HRP/column The sugar binding sites are protected during the conjugation process Operating Flow Rates: Up to 2 mL/min so the Con A activity is well maintained. Figures 2 and 3 show the pH Range: pH 5–8 specificity of the ProSwift ConA-1S column. Three pNP-labeled sugars Operating Temperature: ≤30 °C were separated based on their different affinities towards Con A. Maximum Pressure: 2300 psi Horseradish peroxidase (HRP), which is a glycoprotein with rich Organic Solvent Limit: 10% Methanol high-mannose type glycans, was bound to the Con A column and was eluted with α-methyl-mannopyranoside, which has high affinity for Con A.

2 OptimizationA New Monolithic of the ConA Separation Affinity Power Column of 1-Dfor NanoPurification LC Analysis and Analysisof Proteomics of Glycans, Samples Glycopeptides, and Glycoproteins Loading Capacity of ConA-1S column column ruggedness The loading capacity for HRP is no less than 2.0 mg on the ProSwift The ProSwift ConA-1S column can be regenerated easily by washing ConA-1S column at a flow rate of 1 mL/min. Figure 4 shows the linearity with conditioning buffer after sample binding and elution. The rugged of area to sample load for HRP when loaded onto the ProSwift ConA-1S column chemistry allows hundreds of run cycles with minimal capacity column. This correlation allows the ProSwift ConA-1S to be used for loss. Figure 5 shows the ProSwift ConA-1S column maintains good quantitation of enriched species. capacity after 100 injections and elutions of HRP.

Column: ProSwift ConA-1S (5 × 50 mm) Column: ProSwift ConA-1S (5 × 50 mm) Flow Rate: 1 mL/min Flow Rate: 1.0 mL/min Eluents: A. 50 mM NaOAc, 0.2 M NaCl, pH 5.3, Eluent A: 50 mM NaOAc, 0.2 M NaCl, pH 5.3, 1 mM CaCl , 1 mM MnCl 180 2 2 with 1 mM CaCl2, 1 mM MgCl2 B. 100 mM α-Methyl-mannopyranoside Eluent B: 100 mM α-Methyl-mannoside in eluent A in eluent A 160 Gradient: 0% B for 2 min, 0-100% B in 0.5 min, Gradient: 100% A from 0–2 min, 50% 100% B for 7.5 min B from 2.5–8 min 140 Temperature: 30 °C Temperature: 25 °C Sample: 5–30 mg/mL HRP Column Overloaded Sample: 25 mg/mL HRP 120 Detection: UV 403 nm Detection: UV at 403 nm Inj. Volume: 100 µL Inj. Volume: 100 µL Inj. #101 100 91 mAU 81 80 71 Area (mAu/min) 61 60 51 41 40 31 21 20 11 1 0 00.5 11.5 22.5 33.5 0 1 2 3 4 5 6 7 8 HRP (mg) Minutes 28189 27455

Figure 4. Linearity of area to sample load for HRP on ConA-1S column. Figure 5. One hundred binding-elution cycles of HRP.

3 Column: ProSwift ConA-1S Column: ProSwift ConA-1S

Flow Rate: 1.0 mL/min Eluent A: 50 mM NaOAc, 0.2 M NaCl, pH 5.3, with 1 mM CaCl2, 1 mM MnCl2 Eluent A: 50 mM NaOAc, 0.2 M NaCl, pH 5.3, Eluent B: 125 mM α-Methyl-mannoside and 125 mM -Methyl-glucoside in mobile phase A with 1 mM CaCl2 and 1 mM MnCl2 α Eluent B: 100 mM α-Methyl-mannoside in eluent A Gradient: 100% A from 0–3.5 min, 15% B from 4–18 min Gradient: 0% B for 2 min, 0–100% B in 0.5 min, Flow Rate: 0.5 mL/min 100% B for 3.5 min Temperature: 25 °C Temperature: 25 °C Inj. Volume: 50 µL Sample: 2-AB labeled serum protein N-glycans Detection: UV at 280 nm Detection: Fluorescence, EM at 420 nm Sample: 5 mg/mL Chicken ovalbumin

Unbound fraction reloaded

mAU mV Unbound fraction

Chicken ovalbumin loaded

0 1 2 3 4 5 6 0 2 4 6 8 10 12 14 16 18 Minutes Minutes 27456 27552

Figure 6. Purification of fluorescent-labeled glycans. Figure 7. Separation of chicken ovalbumin glycoforms.

Applications Separation of Glycoprotein Glycoforms Con A is one of the most well characterized and widely used lectins. Protein glycoforms with different affinities to Con A can be separated It binds to b-mannose, and to b-glucose with weaker affinity. Usually on the ProSwift ConA-1S column. In Figure 7, chicken ovalbumin was high-mannose type glycans bind to Con A strongly, and some hybrid fractionated into an unbound and a bound fraction on this column. The type glycans can also bind to Con A with good affinity, while complex unbound fraction was collected and reloaded onto the column. All of the type glycans usually have very weak affinity towards Con A. As previously unbound fraction again eluted in the flow-through. Elution of b-mannose is commonly expressed on most glycoproteins, a the unbound fraction indicates that it has different glycosylation pattens Con A-affinity column is a useful tool for purification and enrichment than the bound fraction. for glycans, glycopeptides, and glycoproteins.

Fractionation of Glycans Glycan samples can be fractionated on the Con A column based on their different affinities to Con A. Figure 6 shows fluorescence-labeled serum N-glycans were fractionated into two fractions on the ProSwift ConA-1S column.

4 OptimizationA New Monolithic of the ConA Separation Affinity Power Column of 1-Dfor NanoPurification LC Analysis and Analysisof Proteomics of Glycans, Samples Glycopeptides, and Glycoproteins Enrichment of Glycopeptides HRP was digested with trypsin. The tryptic digest was fractionated on the ProSwift ConA-1S column. The bound and unbound fractions were collected and analyzed on a reversed-phase column (Figure 8).

A

Column: ProSwift ConA-1S Eluent A: 50 mM NaOAc,

0.2 M NaCl, pH 5.3, with 1 mM CaCl2 Eluent B: 100 mM α-Methyl-mannoside in eluent A Gradient: 100% A from 0–5 min, 100% B from 5.5–15 min Flow Rate: 0.5 mL/min mAU Unbound fraction Bound fraction Temperature: 25 °C Detection: UV at 214 nm Sample: HRP tryptic digest

024 6 8 10 12 14 16 18 20 Minutes

® Column: Acclaim PA2 (3 × 250 mm) Temperature: 30 °C Eluent A: 0.03% TFA in water Detection: UV 214 nm B Eluent B: 0.02% TFA in acetonitrile Gradient: 2–50% B in 120 min Flow Rate: 0.4 mL/min * Total peptide pool * * * * *

ConA-unbound fraction mAU

* * ConA-bound fraction * * * *

0 20 40 60 80 100 120 Minutes 27553

Figure 8. Enrichment of HRP glycopeptides. A) Enrichment of glycopeptides on ProSwift ConA column. B) Profiling of HRP peptide pools on a reversed-phase column.

5 Enrichment of Glycoproteins Depleted human plasma proteins were fractionated on the ProSwift ConA-1S column (Figure 9).

Column: ProSwift ConA-1S (5 × 50 mm) Flow Rate: 0.5 mL/min

Eluent A: 50 mM NaOAc, 0.2 M NaCl, pH 5.3, with 1 mM CaCl2 Eluent B: 100 mM α-Methyl-glucoside in eluent A Gradient: 0% B for 4 min, 0–100% B in 0.5 min, 100% B for 3 min Temperature: 25 °C Sample: Depleted human serum protein Detection: UV 280 nm

mAU

100%

Unbound Bound

0% B 0%

0 2 4 6 8 10 Minutes 27554 Figure 9. Enrichment of glycoproteins.

Conclusion • A novel monolithic HPLC Con A-affinity column with high capacity and specificity was developed. • The ProSwift ConA-1S column provides highly efficient enrichment, purification, and analysis on glycan, glycopeptide, and glycoprotein samples. • The HPLC-compatibility of the ProSwift ConA-1S column allows automation, high throughput, and excellent reproducibility.

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