LudgerT ag™ Procainamide Glycan Labelling System Sensitive profiling of glycan samples using (U)HPLC, ESI-MS, and LC-ESI-MS
In this presentation you will see how: Procainamide is applicable to both (U)HPLC, ESI-MS, and LC-ESI-MS analysis Procainamide labelling is The validation of the suitable for 25pmol – 25nmol LudgerTag procainamide quantity of sample and can labeling system makes it identify minor glycan species suitable to GMP work This technology can be The incorporation of 2PB used for biopharma studies reductant makes the method (e.g. IgG and EPO, N and O- glycosylation) easy/safer to use than sodium and different biological samples cyanoborohydride (e.g. saliva and blood) Procainamide Glycan Labelling | 2
Fluorescent labels and their suitability to (U)HPLC, MS Fluorophore Structure (U)HPLC MS Comments Enhances MS ionization efficiency. Procainamide Suitable for LC-MS systems, HPLC (HILIC, RP, WAX) and ESI- (PROC) ***** ***** MS Low sample amount required Improves identification of very low abundance glycans The most widely used fluorescent tag. 2-aminobenzamide **** **** Suitable for HPLC (HILIC, RP, WAX) and MS (2-AB) Higher sample amount required for ESI-MS Suitable for HPLC (HILIC, RP) and MS 2-aminobenzoic acid **** **** Higher sample amount required for ESI-MS (2-AA) Suitable for HPLC and MS 2-aminopyridine *** **** Higher sample amount required for HPLC and MS analysis (2-AP) 5 stars = excellent, 4 stars = good, 3 stars = fair, 1 - 2 stars = poor Procainamide Glycan Labelling | 3
The anatomy of procainamide 4-amino-N-(2-diethylaminoethyl) benzamide 2-(diethylamino)ethyl group enhances the ionization efficiency in the positive ESI mode Fluorescent group for detection at Ex 310 and Em 370 nm in UHPLC O N Added mass N 219.172 Da H H 2 N Site of attachment to glycan via reductive amination Procainamide Glycan Labelling | 4
LudgerTag Procainamide Labelling Kits Our procainamide labelling technology uses the same reductive amination labelling method that has been used for 2AB & 2AA. The following procainamide labelling kits are available: Kit name Reductant Kit size LT-KPROC-24 24 samples Sodium cyanoborohydride LT-KPROC-96 96 samples LT-KPROC-VP24 2-picoline borane 24 samples Sodium cyanoborohydride is a gold standard reductant used in glycan labelling. Best practice is to perform the labelling in a fume cupboard. 2-picoline borane (2-PB) is less toxic than sodium cyanoborohydride and can be used ‘on a laboratory bench’. Procainamide Glycan Labelling | 5
Standards for procainamide workflow Process controls: Glycoproteins – IgG, Fetuin Unlabelled glycans – including: N- and O-glycans • Purified di-, tri-, tetraantennary glycans • High mannose glycans • Glycan libraries • System suitability standards: Labelled glycans: Purified di-, tri-, tetraantennary glycans • High mannose glycans • Glycan libraries • Labelled Glucose Homopolymer (GHP) For full list of standards check comparison table: https://www.ludger.com/system-suitability-standards Procainamide Glycan Labelling | 6
LudgerTag Procainamide Workflow Released Glycans Procainamide Labelling Enrichment (Purification) and Clean up Ready for Analysis Procainamide Glycan Labelling | 7
How we are using the LudgerT ag Procainamide Labelling System - for N-glycans
Workflow - LudgerTag Procainamide for N-glycan profiling and identification Release Labelling Clean-up N-glycans Procainamide Labelled N-glycans Glycoprotein LZ-rPNGaseF-kit LC-PBM-96 LT-KPROC-VP24 LC-PROC-96 PNGase F Binding Plate* or LudgerClean LT-KPROC-24 PROC Plate *Optional clean-up, or recommended for LT-KPROC-96 optimal results Procainamide Labelling Kits Procainamide Glycan Labelling | 9
Sample preparation within 1 day – N-glycans Timing for processing of 96 samples Clean-up Glycan Release Labelling 1 hr 1 hr 1 hr 1 hr 2 hr Analyse LZ-rPNGaseF-kit LC-PROC-96 LT-KPROC-VP24 LT-KPROC-24 LT-KPROC-96 Hands-on time Incubation time Procainamide Glycan Labelling | 10
Typical Data (U)HPLC profile ESI-MS profile 6.0e7 Intens. 6 x10 1454.70 5.0e7 4 GU 6.00 Intensity 3.8e7 3 2.5e7 2 1.3e7 1 1476.74 1855.38 0.0e0 0 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z 15.0 20.0 25.0 30.0 35.0 37.0 Time Procainamide Glycan Labelling | 11
Comparison of 2-AB and Procainamide Glycoprofiling by (U)HPLC Procainamide labelling of glycans gives comparable results to the gold standard 2-AB labelling of glycans Comparable in quantitation and in reliability, using a 100.00 2 2-AB Waters ACQUITY UPLC Glycan BEH Amide Column (HILIC) 80.00 60.00 2-AB 60.00 4 50.00 Procainamide 3 40.00 Average Rel. % Area 40.00 5 20.00 30.00 1 0.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 20.00 Minutes UPLC Chromatogram of 2-AB labeled glycans from mAb 10.00 280.00 2 0.00 260.00 Procainamide 1 2 3 4 5 240.00 Peak number 220.00 Comparison of Average Relative % Area 200.00 180.00 3500000 160.00 4 2-AB 140.00 3000000 3 120.00 Procainamide 2500000 100.00 80.00 2000000 60.00 5 1500000 40.00 20.00 1 1000000 0.00 500000 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 Minutes 0 UPLC Chromatogram of Procainamide labelled glycans from mAb Comparison of Signal Intensity (FLR) Procainamide Glycan Labelling | 12
Procainamide Glycoprofiling by LC-MS GU 5.66 Intens. Procainamide [mAU] Rel. % Area = 42.85% 8 x10 LC-FLR trace 2 2+ Intens. MS spectra 841.81 8 x10 1 4 2+ 0 2 852.82 Intens. 8 1+ Procainamide x10 1479.62 0 6 ESI-MS trace 400 600 800 1000 1200 1400 1600 1800 m/z 4 2 Intens. 1479.63 5 0 x10 MS/MS spectra 16 18 20 22 24 26 28 30 32 Time [min] 1.5 1.0 1276.56 790.37 587.31 0.5 740.81 991.34 1114.47 441.24 146.07 0.0 200 400 600 800 1000 1200 1400 m/z Glycan composition: H 3 N 4 F 1 Possible structure: FA2 Procainamide Glycan Labelling | 13
Recommend
More recommend
