GlycoWorks Sample Preparation Consumables Glycosylation is one of the most common forms of post-translational modification (PTM) of human and other eukaryotic proteins. Glycosylated proteins (glycolproteins) make up 50-70% of human proteins. Consequently, glycans play a critical role in a myriad of physiological and pathological reactions ranging from immunity, to blood clotting, to cell development, and cell death. Glycoprotein analysis involves identifying complex N- and O-Linked structures composed of frequently similar and repeating sugar moieties. Hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection is a well-recognized and reliable technique that effectively separates and quantitates isolated glycans after their derivatization with fluorescent labels. Waters now offers the sample preparation aspect of the Glycan Application Solution to help users get the correct answers faster and more reliably: ■■ Versatile offering for for individual samples up to high-throughput analyses ■■ Convenient, easy to use methodologies that bring a user through sample preparation to data collection and interpretation ■■ Seamless integration with Waters ACQUITY UPLC® and related column chemistries, and informatics solutions for data analysis. SAM P LE P REPA RAT ION GlycoWorks High-throughput Prep Kit 16.0 Waters GlycoWorks Consumables offer a more convenient, versatile, comprehensive and effective sample preparation solution for glycan analysis. ■■ ■■ 12.0 ■■ Expanded line of consumables that includes labeling kits and standards 8.0 2 4.0 7 3 2.0 9 4 10 0.0 2 Updated support documentation for use with UV/FLR/MS and MALDI Versatile formats for high throughput and single use device users 6 6.0 Optimized HILIC Sorbent for high recovery of N-glycans and for effective labeling reaction clean up prior to LC/ FLR/ MS analysis. ■■ 8 10.0 EU RapiGest™ for solubilization of the glycoprotein and efficient PNGase F enzymatic digestion of the N-linked glycans 5 14.0 4 6 8 10 12 Min Retention Time (min) 30 n=3 25 Relative Peak Area (%) ■■ Human Serum IgG ~10 µg starting material 1 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 Common Peak Number Structure 1 FA2 G0F 2 A2[6]G1 G1a 3 A2[3]G1 G1b 4 A2BG1 G1F+GN 5 FA2G1 G1Fa Denaturation with MassPREP RapiGest DTT Reduction (if necessary), Deglycolisation by PNGase F 6 FA2G1 Peak 7 FA2BG1 G1F+GN 8 FA2G2 G2F 2 Protein Removal GlycoWorks HILIC µElution Plate 9 FA2BG2 G2F+GN 10 FA2G2S1 G2F+SA 3 2-AB Labeling of Purified Glycans 4 Excess Labelling Regent Removal GlycoWorks HILIC µElution Plate Sample Preparation from Glycoprotein to Purified 2-AB Labeled Glycans Glycoproteins 1 Purified 2-AB Labeled Glycans MALDI MS Reproducibility using HILIC. 3 separate sample preps were performed and relative recovery was analyzed of the 10 peaks annotated in the above chromatogram. Typical Results of IgG Glycan Release using the GlycoWorks HILIC Sorbent with GlycoWorks Control Standard and 2AB Labeling Kit LC-FLR 2 ADDIT IONAL CONSUMABL ES Dextran Calibration Ladder 2AB-Dextran Ladder, FLR GU 15 GU 10 FLR λex = 330 nm λem= 420 nm GU 5 GU 2 T he Dextran Calibration Ladder allows the user to tie in the entire GlycoWorks Sample Prep solution seamlessly to the Waters U ACQUITY PLC System and GlycoBase Database Search. Using this 2-AB Labeled standard allows the user to calibrate their system based on GU units and have confidence in results. The lyophilized 2AB Dextran Calibration Standard, 200 µg, was reconstituted in 60% Acetonitrile, and the injection volume was 2 µl. Glycan Performance Standard The Glycan Performance Test Standard is 2-AB labeled Humanlike IgG and is QC verified to contain the components needed to benchmark and evaluate ACQUITY UPLC BEH Glycan, 1.7 µm Columns. It is also valuable to use as an additional 2-AB labeled control to assess digestion and labeling reaction efficiencies. 6 2 11 7 3 1 10 12 14 Peak Identification 1. G0 (NGA2) 2. G0F (NGA2F) 3. Man-5 4. G0F+GN 16 4 13 89 5 18 5. G1 6. G1Fa (NA2G1F) 7. G1Fb (NA2G1F) 8. G1F+GN 10 20 12 22 24 9. Man-6 10. G2 (NA2) 11. G2F (NA2F) 12. G1F+SA 26 28 30 min 13. G2F+SA (A1F) 3 CH ROMATOGRA PHIC ANALYSIS Waters ACQUITY UPLC BEH Glycan Column was specifically developed and is QC tested to provide superior UPLC resolution in less time for a range of glycan structures. When coupled with Waters ACQUITY UPLC System, the column chemistry is ideally suited to help users get the correct answers faster. ■■ Improved component resolution in less time compared to existing HPLC-based methods ■■ Optimized for use with ACQUITY UPLC System with fluorescence detection ■■ Based on Waters BEH particle and binding technology for stable and reproducible labeled glycan structures ■■ Quality control tested with Glycan Performance Test Standard for consistent batch-to-batch reproducibility. 3 2 4 6 2.1 x 150 mm 11 3 1 7 4 89 5 13 12 10 3 4 2.1 x 100 mm 3 4 2.1 x 50 mm 0 5 10 15 20 25 30 35 40 min Column Lengths that Satisfy Resolution and Sample Throughput Requirements ACQUITY UPLC BEH Glycan columns are available in three column lengths for various application needs that balance resolution, speed of analysis, and solvent consumption. Shown here are three separations of the same 2-AB labeled IgG glycan sample noting comparative Rs for respective peaks 3 and 4 from each analysis. 4 GLYCAN ANALYSIS WORK FLOW Sample Preparation: Chromatographic Analysis: Data Analysis and Interpretation: GlycoWorks line of sample preparation products and Additional Support Consumable Standards and Kits ACQUITY UPLC System with FLR detection and ACQUITY UPLC BEH Glycan Columns Coupled with Empower ® or UNIFI ® to acquire and process data. ™ ■■ ■■ Efficient methodology using optimized HILIC sorbent for high recovery of N-linked glycans. ■■ Improved component resolution in less time compared to existing HPLC-based methods ■■ Utilize the Waters’ UPLC Database within NIBRT’s GlycoBase 3.0+ . ■■ This UPLC database was developed by the Waters-NIBRT collaboration. Quantitative information provided for a range of glycans. 2AB Dextran Standard (GU) GlycoWorks™ Sample Prep Consumables HILIC UPLC-FLR Empower 3.0 UNIFI 1.6 15000.000 12500.000 EU x 10e4 Fluorescence Released 2AB Glycans from Biotherapeutic 10000.000 7500.000 5000.000 2500.000 0.000 Min. 5 10 15 20 25 30 35 HPLC F(3)XM3 A3 F(6)A2 Galβ1-4[Fuca1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-6[Galβ1-3]GalNAc avg.: 1189.080 mono: 1188.428 avg.: 1520.402 mono: 1519.566 avg.: 1463.351 mono: 1462.544 avg.: 1260.158 mono: 1259.465 UPLC 5.89 ±0.029 5.88 ±0.045 5.90 ±0.029 5.88 ±0.032 5.90 ±0.042 GU Search 5.93 The complete Glycan Application Solution includes: Waters ACQUITY UPLC H-Class, Xevo® G2-S Qtof, UNIFI, a Glycan column and the Glycan Performance Standard. The release of UNIFI 1.7 is currently scheduled for Q3 2013. Waters Glycan Analysis Work Flow 5 ORDERING INFORMAT ION Description Part Number Dextran Calibration Ladder 186006841 ACQUITY UPLC BEH Glycan 1.7 µm 2.1 x 50 mm 186004740 ACQUITY UPLC BEH Glycan 1.7 µm 2.1 x 100 mm 186004741 ACQUITY UPLC BEH Glycan 1.7 µm 2.1 x 150 mm 186004742 ACQUITY UPLC BEH Glycan, VanGuard 3-pack 186004739 Glycan Performance Test Standard 186006349 GlycoWorks Reagent Kit 186007034 GlycoWorks High-throughput Prep Kit 176003090 GlycoWorks Single Use Prep Kit 176003119 ™ Check www.waters.com/standards for updated information. ©2013. Waters, ACQUIT Y UPLC, UPLC, Empower and UNIFI are registered trademarks of Waters Corporation. GlycoWorks, RapiGest , VanGuard, and T he Science of W hat’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. ©2013 Waters Corporation. Produced in the U.S.A. March 2013 720004584EN IH-PDF Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com