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Ultra-high throughput, ultrafiltration-based N-glycomics platform for ultra-performance liquid chromatography (ULTRA3) Henning Stöckmann*, Rebecca M. Duke*, Silvia Millán Martín* and Pauline M. Rudd* *NIBRT GlycoScience Group, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount, Merrion, Blackrock, Co. Dublin, Ireland. E-mail:
[email protected] Fax: +353 (0)1 215 8166 ABSTRACT: Accurate, reproducible and fast quantification of N-glycans is crucial not only for the development and quality control of modern glycosylated biopharmaceuticals, but also in clinical biomarker discovery. Several methods exist for fluorescent labeling of N-glycans and subsequent chromatographic separation and quantification. However, the methods can be complex, lengthy and expensive. Here we report an automated ultrafiltration-based N-glycoanalytical workflow combined with a glycan labeling strategy that is based on the reaction of glycosylamines with fluorescent carbamate. The entire protocol is quick, simple, and cost-effective and requires less than 1 µg of protein per sample. As many as 768 affinity purified IgG glycoprotein samples can be prepared in a single run with a liquid handling platform.
INTRODUCTION Modern glycoanalytical and glycomics workflows rely on the accurate quantification of glycans from biological samples. Quantification is typically achieved by labeling the glycans with fluorophores and their subsequent chromatographic analysis with fluorescence detection, which is often used in conjunction with complementary techniques such as mass spectrometry (MS).1,2 By far the most widely used glycan labeling method has been reductive amination with aminobenzene derivatives such as 2-aminobenzamide (2-AB) or 2aminobenzoic acid (2-AA).3 N-Glycan fluorescence labeling with 2-AB has been used for characterizing the N-glycans of many biopharmaceuticals 4 as well as profiling clinical samples.5-7 We have recently developed the first fully automated, costeffective N-glycomics platform.2 The fluorescent N-glycans are prepared from complex biological samples (e.g. serum) on a commercial liquid handling robot. The main purpose of the platform is accurate quantification of N-glycans. The platform is versatile and can be used to determine the glycosylation pattern of individual glycoproteins or classes of glycoproteins, such as immunoglobulin G (IgG). This robotic method showed excellent reproducibility and samples prepared on different days have coefficients of variation (CV’s) that are typically below 10% (peaks with a relative percentage area of 2 mm Hg vacuum, 30 min). Denaturation buffer (25 μL per well, 100 mM sodium bicarbonate, 50 mM dithiothreitol (DTT), 0.1% sodium dodecyl sulfate (SDS)) was dispensed into 2 × 384 well ultrafiltration plates. After 10 minutes incubation at room temperature, the samples were mixed 10 times (mixing volume: 15 μL, flow rate: 10 μL/s) and transferred to a 384-well PCR plate (Armadillo). The plate was placed into a robotic incubation chamber at 95°C for 10 minutes. The plate was
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removed from the incubator and equilibrated to room temperature for 10 minutes. 1M iodoacetamide (IAA), 10 μL, was dispensed into each well of the ultrafiltration plate and the samples (25 µl) were transferred back into the 384 well PCR plate. The samples were mixed 5 times (mixing volume 20 µL, flow rate, 10 μL/s). After 10 minutes incubation at room temperature, the ultrafiltration plate was stacked onto a 240 µL collection plate (Corning block) and centrifuged (3700g, 30 min, room temperature). Next, 10 μL of a 25 mM sodium bicarbonate solution was dispensed into each well and the ultrafiltration plate was centrifuged (3700g, 30 min, room temperature). The deglycosylation mix, 12 µL (0.4 µl of PNGase F (2.5 U/mL), 25 mM sodium bicarbonate) was dispensed into each well and the plate was then covered with a lid and incubated on a robotic orbital shaker (shaking orbit: 2 mm, shaking speed: 700 rpm, temperature: 38 °C, incubation time: 30 min). The ultrafiltration plate was stacked onto a 30 μL/well PCR plate (Armadillo) and centrifuged (3700g, 10 min, room temperature). Finally, 10 μL of a 25 mM sodium bicarbonate solution was dispensed into each well of the ultrafiltration plate (still stacked onto the PCR plate) and the assembly was centrifuged (3700g, 10 min, room temperature). For glycan labeling, 5 μL of glycan sample (PCR plate) was transferred to a 95 µL Corning block and 11.6 μL of AQC (3 mg/mL MeCN) was added. 3 μL of this crude mixture was directly injected into the UPLC system. Alternatively, after the PNGase F release, glycans can be frozen and are stable at -20 °C for labeling at a later date. Automated 96 Well Glycan Solid Phase Extraction (SPE) using Hypersep Diol Cartridges (Optional). Labeled samples (16.6 μL 30:70 Buffer:MeCN) were transferred from the 384 to a 96 well plate. The volume was adjusted to 1 mL of MeCN/H2O (95:5) and SPE was carried out according to the previously reported protocol.2 Ultra Performance Liquid Chromatography (UPLC) with Fluorescence Detection (FLD). Separation of AQCderivatized N-glycans was carried out by UPLC with fluorescence detection on a Waters ACQUITY UPLC H-Class instrument consisting of a binary solvent manager, sample manager, and fluorescence detector under the control of Empower 3 software (Waters, Milford, MA, USA). The HILIC separations were performed using a Waters Ethylene Bridged Hybrid (BEH) Glycan column (150 × 2.1 mm i.d., 1.7 μm particles) with 50 mM ammonium formate (pH 4.4) as solvent A and MeCN as solvent B. The column was fitted with an ACQUITY in-line 0.2 µm filter. The separation was performed using a linear gradient of 70−53% MeCN at 0.56 mL/min in 16.5 min for IgG N-glycan separation. An injection volume of 3 μL prepared in 70% v/v MeCN was used throughout. Samples were maintained at 5°C prior to injection, and the separation temperature was 40°C. The FLD excitation/emission wavelengths were λex = 245 nm and λem= 395 nm, respectively. The system was calibrated using an external standard of hydrolyzed and 2-AB-labeled glucose oligomers to create a dextran ladder, as described previously. A fifth-order polynomial distribution curve was fitted to the dextran ladder to assign glucose unit (GU) values from retention times (using Empower software from Waters). Liquid Chromatography-Mass Spectrometry-Fluorescence Detection (LC-MS-FLD). LC-MS was performed using an ACQUITY UPLC system equipped with a BEH Glycan column (150 × 1.0 mm i.d., 1.7 μm particles), which was coupled to a Waters Xevo G2 QTof system. For MS acquisition data
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the instrument was operated in positive-sensitivity mode with a capillary voltage of 2.3kV. The ion source block and nitrogen desolvation gas temperatures were set at 120°C and 400°C, respectively. The desolvation gas was set to a flow rate of 600 L/h. The cone voltage was maintained at 20V. Fullscan data for IgG N-glycans were acquired over m/z range of 450 to 2500, but this range may be extended. Data collection and processing were controlled by MassLynx 4.1 software (Waters, Milford, MA, USA). The FLD excitation/emission wavelengths were λex = 245 nm and λem= 395 nm, respectively; data rate was 1pts/second and a PMT gain = 10. Sample injection volume was 8 μL (75% MeCN). The flow rate was 0.150 mL/min and column temperature was maintained at 60°C; solvent A was 50 mM ammonium formate (pH 4.4) and solvent B was MeCN. A 40 minute linear gradient was used and was as follows: 28% A for 1 min, 28-43% A for 30 minutes, 43-70% A for 1 minute, 70% A for 3 min, 70-28% solvent A for 1 min and finally 28% A for 4 minutes. Synthesis of 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate, AQC (1). 560 mg (2.18 mmol) of N,N’disuccinimidyl carbonate, was dissolved in 20 mL of MeCN in a round-bottom flask and the solution was stirred at RT. When the solid had dissolved (slight warming may be required), 270 mg (1.87 mmol) of 6-aminoquinoline was added and the reaction mixture was stirred at ambient temperature for 3 h. The precipitate was collected by vacuum filtration and the product dried under vacuum to give 350 mg of a pale yellow solid. Exoglycosidase Digestions. After removal of excess AQC label using HypersepDiol, equal aliquots of IgG N-glycans were dried in a vacuum centrifuge and then digested according to the sequence shown in Figure 3. For enzymes ABS, BTG, GUH, BKF and JBM, 1, 2, 2, 4 and 4 µl of each was used per digestion, respectively. All digestions were carried out in a final volume of 10 µl, at 50 mM NaOAc pH 5.5 for 42h. RESULTS AND DISCUSSION Fundamental to our newly streamlined glycoanalytical platform is the revised synthesis of the glycosylamine-reactive fluorophore (1), AQC. The synthesis was previously reported in the literature;14 however, an alternative shorter and more convenient synthesis is reported in the experimental section of this paper. The synthesis is reliable and does not require specialist organic synthesis equipment or skill. The proposed mechanism for the formation of AQC labeled N-glycans is shown in Scheme 1. The 384 well plate workflow detailed herein was adapted from our recently published paper2 and the 2-AB glycan labeling step was replaced with the AQC labeling reaction. The previous method included a glycan purification step, whereby after deglycosylation, the N-glycans were extracted from solution using a solid supported hydrazide resin. 2,15 During method development, it became apparent that this step was not required for AQC labeling of IgG N-glycans. Hence, while it took 22 h to process 96 human serum IgG samples in our previously reported method, the processing time for the same number of samples can be reduced by more than 50% using the ULTRA3 assay.
Scheme 1: Labeling of a representative glycosylamine (2) with AQC (1) to give the fluorescent urea derivative (3). AQC can hydrolyse in the presence of water to give (4) and (5) 6-aminoquinoline (AMQ). Compound (4) can further decompose to give (6) N-hydroxysuccinimide (NHS), and carbon dioxide, CO2.14 An overview of the ULTRA3 assay is shown in Scheme 2. The workflow is initiated by an automated affinity purification step of the glycoprotein of interest, e.g. isolation of IgG from serum using Protein G.16 The Protein G affinity purification of IgG is not shown in the scheme but was detailed in our previous publication.2 The proteins are denatured, washed and the N-glycans are cleaved from the protein by the enzyme Peptide-N-Glycosidase F (PNGase F). The N-glycans are then separated from the protein by ultrafiltration (UF) (Steps 1-8). These steps are conveniently carried out on the same 384 well UF plate, with vacuum filtration performed by the Hamilton robot. Residual impurities and buffer salts do not interfere with the subsequent AQC labelling reaction (Step 9). For step 9, the entire filtrate consisting of glycosylamines (ca. 15 µl) can be labeled with AQC, or alternatively, an aliquot (e.g. 5 µl) can be removed for manual or robotic labeling in a 384 well plate. The optional 96 well SPE step detailed in the experimental section is also not depicted in Scheme 2. This step is not required for N-glycan analysis of IgG using the ULTRA3 assay and its removal considerably reduces the sample processing time (by ca. 4-6 h). The resulting AQC labeled N-glycan profile of IgG is shown in Figure 1 (A). For the sake of comparison, the 2-AB labeled profile is shown in Figure 1(B). The AQC and 2-AB N-glycan profiles were generated from the same IgG sample using the same quantity of unlabeled N-glycans. For the 2-AB sample, after step 8, additional wash steps were carried out (due to the sensitivity to salt and detergent) and the glycosylamines were then reduced and labeled with 2-AB (2 h, 65°C). After labeling, the excess 2-AB was removed from the sample using SPE (Hypersep Diol). Figure 1 shows that the N-glycan profiles are similar, with slight differences in the resolution of selected peaks, as anticipated.
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Scheme 2: Automated sample preparation workflow, consisting of sample preparation (1), protein denaturation and cleanup (2-6), enzymatic N-glycan release (7), N-glycan collection (8), AQC labeling (9) and UPLC analysis. . However, in comparison to the AQC profile, the fluorescence emission of the 2-AB profile is about 30-fold less intense (see emission units (EU) on y-axis). , This indicates that the ULTRA3 assay, combined with AQC labeling, is suitable for analyzing and profiling IgG samples for which only a minimal amount of material is available.
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Although not required for the ULTRA3 assay, we investigated the profiles of AQC labelled IgG N-glycans before and after SPE (Hypersep Diol), Figure 2. The chromatograms clearly show that the dye peak (ca. 0 – 2 min) does not interfere with the N-glycan profile (-SPE) as the first bi-antennary structure
(A2) elutes at ca. 5 minutes. Integration of the peaks in each IgG N-glycan profile, pre- and post SPE, and subsequent comparison of the relative percentage peak areas showed that a slight selectivity was introduced to the N-glycan profile by the SPE step, although SPE resulted in full glycan recovery. The selectivity is clearly visible in the overlaid chromatograms in Figure 2 and the encircled peaks show the loss of sialylated Nglycans, ca. 16-18%. Compared to many other fluorescent Nglycan derivatization reagents (e.g. 2-AB, 2-AA), the effectiveness of the enrichment and purification of any SPE resin (e.g. the final yield, sample matrix effects and selectivity) can easily be evaluated with AQC labeled N-glycans. It is possible to analyse the crude labeled N-glycan sample using UPLC and then compare the profiles after SPE. Hence, despite the previously demonstrated high yield and reproducibility of the HyperSep Diol SPE step, if possible, in order to enhance the success of glycan biomarker discovery and to carry out accurate relative quantification of N-glycans, the Hypersepdiol SPE step is best avoided for UPLC-FLR detection.2 Often the lack of sample clean-up may cause concern with regard to the longevity of the UPLC column. However, despite the absence of the SPE step in this method, up to 2000 injections were carried out on the same UPLC HILIC column without any observable effects on the resolution of the N-glycan profiles. After isolation of the N-glycans (step 8, Scheme 2), the samples may be stored at -20°C and labeling can be carried out at a later stage. We have also used AQC to label the N-glycans from more complex sample matrices, e.g. samples that have not been affinity purified, such as cells and tissue. Often for these samples, SPE is required directly after labeling to remove not only excess label, but also non-glycan analytes. When such complex samples were frozen and then thawed, in the presence of excess AQC label, a precipitate was observed. Hence, in this instance it is advised to store the samples as unlabeled N-glycans or labeled glycans post SPE.
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Figure 2: UPLC chromatograms of AQC labeled IgG N-glycans before and after SPE using Hypersepdiol resin.
EXOGLYCOSIDASE DIGESTIONS N-glycans are routinely characterised using exoglycosidase enzymes.17,18 A range of exoglycosidase enzymes are commercially available, with both broad and narrow specificities (NEB and Prozyme), and these can be used to digest all of the N-glycan structures from an IgG sample back to one common core structure, mannose 3 (M3). Firstly, the labeled IgG glycan sample is split into equal aliquots: one sample to act as an undigested control (UND) and the remaining aliquots for sequential digestions. Terminal sialic acids must first be removed from the non-reducing end of the N-glycans using a sialidase enzyme (e.g. Arthrobacter Sialidase, ABS). Fucosidase, such as Bovine Kidney Fucosidase (BKF), is usually the next enzyme in the sequence. This removes any fucose residues in an α(1,6) linkage on the chitobiose core of the IgG N-glycans, as well as selected fucose residues on the outer arms of the glycan structure. This digestion is followed by removal of β(1,4) linked galactose residues (e.g. using Bovine Testis Galactosidase (BTG)) and then finally the Nacetylglucosamine residues (β(1,2) linked) are removed using a hexosaminidase (e.g. from Streptococcus pneumonia, GUH). Due to the presence of bisected N-glycans on human serum IgG, small quantities of N-glycans remain only ‘partially’ digested at the end of the exoglycosidase panel. In a similar manner to the sequencing of 2-AB labeled Nglycans, we have evaluated the above exoglycosidase enzymes for the digestion of AQC labeled N-glycans. With some minor changes to the enzyme sequence, we have shown that AQC labeled N-glycans can be successfully sequenced using these enzymes. The combination of enzymes used for the digestion is shown in Figure 3. During our investigations difficulty removing core fucose from AQC labeled N-glycans was observed (using 1 µl of BKF in 10 µl of buffered sample). This was possibly due to steric hindrance, i.e. the close proximity of the α-fucose residue to the AQC label (which has a higher molecular mass than 2-AB). Hence, the presence of undigested material was overcome by using a higher concentration of BKF (4 µl of enzyme in 10 µl), digestion for a longer period of time (48 h) and also by adding BKF at the end of the panel, so that all other exoglycosidase enzymes are present to aid
digestion (see (5) in Figure 3 and Figure S-1 in the SI). This also resulted in a more cost efficient digestion panel as opposed to adding 4 µl of BKF to 3 separate digestions. As anticipated, there were bisected N-glycans in the final digestion of AQC labelled N-glycans, see Figure 3 (5). (1) UND
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Figure 3: Sequencing of AQC labeled IgG N-glycans using exoglycosidase enzymes. Only the major glycan structures are shown in the undigested profile (UND). See Figures S-4 and 5 of the SI for a description of enzyme specificities, an explanation of Oxford glycan nomenclature and Table 1 for a list of the N-glycans identi-
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fied in human serum IgG using the ULTRA3 assay and the above digestion panel.
The most abundant N-glycan, M3, in the final digestion, can be further digested back to M1 using Jack Bean Mannosidase (JBM), see Figures S-2 and 3 of the SI. This digestion also indicated the presence of low quantities of high mannose Nglycans in the human serum IgG sample. A 2-AB labeled dextran ladder (glucose homopolymer) can be used to assign GU values to glycan profiles. The ladder is used to assign Glucose Unit (GU) values to each of the peaks. Peak annotation of the chromatograms is conducted using N-glycan sequencing and reference data stored in a dedicated database, GlycoBase 3+ (glycobase.nibrt.ie).19 The same ladder can also be used to assign GU values to AQC labeled IgG N-glycans. Although these are clearly not the same values that would be derived for 2-AB labeled N-glycans (and hence not compatible with Glycobase 3+), the values assist in structure assignment. For example, the GU value of a fucosylated glycan peak (2AB) is expected to shift by ca. 0.3 0.4 GU after digestion with BKF. Figure 3 (5) shows that similar shifts were observed for AQC labeled N-glycans, (see (4) to (5), for which FM3 shifted from 5.15 to M3 at 4.78 GU). Due to their structural complexity, N-glycans are routinely characterised using more than one analytical method (i.e. 2D analysis). LC-MS1 was used to confirm the N-glycan structures assigned by the exoglycosidase digestions. UPLC-MSFLD analysis of AQC labeled IgG N-glycans was carried out using HILIC separation, in both positive and negative modes of ionisation. The results indicated that all AQC labeled Nglycans ionised best in positive mode, for which the [M+2H]2+ ion was usually the most abundant. Figure S-6 of SI shows that the base peak chromatogram (BPI) mirrors that of the FLD trace, however, the sialylated N-glycans (ca. 15 and 18 minutes) clearly do no ionise as efficiently as the neutral Nglycans. Waters Corporation have recently described a synthetically modified AQC based dye (tertiary amine) which can be used for glycan analysis using LC-MS.20 METHOD VERIFICATION After completing the structure assignments (see Figure S-7 and Table S-1 of SI), we compared the relative percentage peak areas of the 2-AB and AQC N-glycan profiles. The comparison showed that similar results were achieved with both labeling strategies, further validating the ULTRA3 assay for high throughput IgG N-glycan profiling, Figure 4. The day to day reproducibility of the ULTRA 3 assay was investigated by analysing the same batch of human serum IgG (900 µg/mL) over a three day period. The studies were carried out using 70 µl of protein solution, i.e. 63 µg of IgG glycoprotein. Two 384 well plates (Plate A and B), with 8 samples in each plate, were processed on three separate days, Day 1-3. Each of the 16 IgG N-glycan profiles were integrated using Empower 3 software and the relative percentage areas of the 23 peaks were compared. The peak areas for the 16 samples (average of Plate A and B replicates) on each day are represented in Figure 5. Excellent %CV’s were observed for the Nglycan peaks, with average values for Day 1, 2 and 3 of 5.5, 4.8 and 2.8%, respectively. The average %CV for each peak for Days 1-3 were calculated and the values showed improve-
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ment compared to our previously published method (see Figure S-8 of the SI) The quantity of IgG that can be processed using the ULTRA3 assay was also investigated. The volume of sample processed through the method was sequentially decreased from 70 to 10 µl, after which 5 and 1 µl of glycoprotein solution were analysed. The results are shown in Figure 5 and indicated that similar peak ratios were obtained for each quantity of IgG glycoprotein analysed.
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Figure 4: Doughnut plot showing the relative percentage peak areas of IgG N-glycans labeled with 2-AB (outer circle) and AQC (inner circle). Due to slight differences in resolution, and in order to compare the glycan profiles, the areas for selected structures were added. Only the major N-glycan structures are shown (Oxford nomenclature). The insert shows the AQC labeled N-glycan profile of IgG. Dotted lines trace selected peaks to the corresponding sections of the plot.
CONCLUSION The maximum throughput of the ULTRA3 assay is very high with the possibility to process 768 IgG samples using two 384 well plates in parallel. The throughput of our previous automated assay was increased significantly from 96 to 768 samples in a single robotic run. Hence, over 8 times more samples can be processed in an even shorter timeframe. The ULTRA3 assay is also more cost effective than the previously reported, with a reduction in the cost of processing one sample by more than 80%. Preliminary data has also shown that the ULTRA3 assay can be used to profile the N-glycans of proteins with glycosylation that is more complex than that of IgG. N-glycan profiling of alpha-1-acid glycoprotein, A1GP, which contains large tetra-antennary glycans as well as more extensive sialylation, was also carried out and showed comparable results to previously reported 2-AB N-glycan profiles (see Figure S-9 of SI).21 Finally, the robotic platform has a highly desirable flexibility and can be used to monitor the glycosylation of important biological glycoproteins other than IgG. Affinity purification has also been investigated using custom made
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Relative Percentage Peak Area
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Peak Number 0.9 ug/1 uL 4.5 ug/ 5 uL 9 ug/ 10 uL 18 ug/ 20 uL 27 ug/ 30 uL 36 ug/ 40 uL 45 ug/ 50 uL 54 ug/ 60 uL 63 ug/ 70 uL
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Figure 5: (A) Relative percentage areas of the 23 peaks in the N-glycan profile of IgG. 8 samples were prepared in both 384 well plates (i.e. 16 samples in parallel) on three different days and analysed by UPLC. (B) Relative percentage area of the 23 peaks in the N-glycan profile of AQC labeled IgG N-glycans. Varying quantities of IgG were processed in parallel using the ULTRA3 assay (63 – 0.9 µg/µL).
robotic tips as opposed to 96 well plates. Tips with immobilised antibodies for IgG isoforms, such as IgG1, IgM and IgA have also been implemented and used to carry out N-glycan profiling.22 Although we have demonstrated here that our method excels at profiling and characterizing a wide range of glycans, migration of O-acetyl groups, such as those found on sialic acids,23-25 may occur under the conditions of release and AQC-labelling. The possibility to use the platform for phosphorylated26-28 and sulfated glycans29-30 will be reported in a separate manuscript. Overall, the methods described here should greatly facilitate the setting-up of an N-glycan profiling and sequencing system as a facility in centres where screening for industrial or clinical applications with specific N-glycan targets exists
ASSOCIATED CONTENT Supporting Information (SI)
Supporting Information Available as notes in text: This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected]. Tel.: +353 (0)1 215 8100 fax: +353 (0)1 215 8166
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT We thank Ciara McManus, Roisin O'Flaherty, Simone Albrecht, Radka Saldova and Rita Dempsey for their help with the preparation of the manuscript. We acknowledge the EU FP7 program HighGlycan, Grant No. 278535 and L’Oreal Paris for funding this work.
REFERENCES
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