GlycoWorks Sample Preparation Consumables

Transcription

1 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 0-70% 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.

2 SAMPLE PREPARATION GlycoWorks High-throughput Prep Kit Waters GlycoWorks Consumables offer a more convenient, versatile, comprehensive and effective sample preparation Human Serum IgG ~0 µg starting material solution for glycan analysis RapiGest for solubilization of the glycoprotein and efficient PNGase F enzymatic digestion of the N-linked glycans EU Optimized HILIC Sorbent for high recovery of N-glycans and for effective labeling reaction clean up prior to LC/ FLR/ MS analysis. 0.0 Updated support documentation for use with UV/FLR/MS Min and MALDI Versatile formats for high throughput and single use device users 0 Retention Time (min) n= Expanded line of consumables that includes labeling kits and standards Relative Peak Area (%) Common Peak Number Structure Sample Preparation from Glycoprotein to Purified -AB Labeled Glycans FA G0F A[6]G Ga A[]G Gb Glycoproteins ABG GF+GN Denaturation with MassPREP RapiGest DTT Reduction (if necessary), Deglycolisation by PNGase F Protein Removal GlycoWorks HILIC µelution Plate FAG GFa 6 FAG Peak 7 FABG GF+GN 8 FAG GF 9 FABG GF+GN 0 FAGS GF+SA -AB Labeling of Purified Glycans Excess Labelling Regent Removal GlycoWorks HILIC µelution Plate Reproducibility using HILIC. separate sample preps were performed and relative recovery was analyzed of the 0 peaks annotated in the above chromatogram. Purified -AB Labeled Glycans Typical Results of IgG Glycan Release using the GlycoWorks HILIC Sorbent with GlycoWorks Control Standard and AB Labeling Kit MALDI MS LC-FLR

3 ADDITIONAL CONSUMABLES Dextran Calibration Ladder The 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 -AB Labeled standard allows the user to calibrate their system based on GU units and have confidence in results. AB-Dextran Ladder, FLR GU GU GU 0 FLR λex = 0 nm λem= 0 nm GU The lyophilized AB Dextran Calibration Standard, 00 µg, was reconstituted in 60% Acetonitrile, and the injection volume was µl. Glycan Performance Standard The Glycan Performance Test Standard is -AB labeled Humanlike IgG and is QC verified to contain the components needed to benchmark and evaluate ACQUITY UPLC BEH Glycan,.7 µm Columns. It is also valuable to use as an additional -AB labeled control to assess digestion and labeling reaction efficiencies min Peak Identification. G0 (NGA). G0F (NGAF). Man-. G0F+GN. G 6. GFa (NAGF) 7. GFb (NAGF) 8. GF+GN 9. Man-6 0. G (NA). GF (NAF). GF+SA. GF+SA (AF)

4 CHROMATOGRAPHIC 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. 6. x 0 mm x 00 mm. x 0 mm 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 -AB labeled IgG glycan sample noting comparative Rs for respective peaks and from each analysis.

5 GLYCAN ANALYSIS WORKFLOW Sample Preparation: GlycoWorks line of sample preparation products and Additional Support Consumable Standards and Kits Efficient methodology using optimized HILIC sorbent for high recovery of N-linked glycans. Chromatographic Analysis: ACQUITY UPLC System with FLR detection and ACQUITY UPLC BEH Glycan Columns Improved component resolution in less time compared to existing HPLC-based methods Quantitative information provided for a range of glycans. Data Analysis and Interpretation: Coupled with Empower or UNIFI to acquire and process data. Utilize the Waters UPLC Database within NIBRT s GlycoBase.0+. This UPLC database was developed by the Waters-NIBRT collaboration. AB Dextran Standard (GU) GlycoWorks Sample Prep Consumables HILIC UPLC-FLR Fluorescence EU x 0e Released AB Glycans from Biotherapeutic Empower.0 UNIFI Min. HPLC UPLC F()XM A ± ± ±0.09 GU Search F(6)A ± ±0.0 Galβ-[Fuca-]GlcNAcβ-Galβ-GlcNAcβ-6[Galβ-]GalNAc The complete Glycan Application Solution includes: Waters ACQUITY UPLC H-Class, Xevo G-S Qtof, UNIFI, a Glycan column and the Glycan Performance Standard. The release of UNIFI.7 is currently scheduled for Q 0. Waters Glycan Analysis Work Flow

6 ORDERING INFORMATION Description Part Number Dextran Calibration Ladder ACQUITY UPLC BEH Glycan.7 µm. x 0 mm ACQUITY UPLC BEH Glycan.7 µm. x 00 mm ACQUITY UPLC BEH Glycan.7 µm. x 0 mm ACQUITY UPLC BEH Glycan, VanGuard -pack Glycan Performance Test Standard GlycoWorks Reagent Kit GlycoWorks High-throughput Prep Kit GlycoWorks Single Use Prep Kit Check for updated information. 0. Waters, ACQUIT Y UPLC, UPLC, Empower and UNIFI are registered trademarks of Waters Corporation. GlycoWorks, RapiGest, VanGuard, and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 0 Waters Corporation. Produced in the U.S.A. March EN IH-PDF Waters Corporation Maple Street Milford, MA 077 U.S.A. T: F: