Barry Boyes 1,2, Shujuan Tao 2, and Ron Orlando 2 1 Advanced Materials Technology, Inc. Wilmington, DE USA 2 Complex Carbohydrate Research Center University of Georgia, Athens, GA USA bboyes@advanced-materials-tech.com
Agenda Protein N-glycan analysis by release, labeling and HILIC General workflow Fused-Core Particles for highly efficient separations New polar bonded phase for HILIC Analysis of N-Glycans by Penta-HILIC Standards and separation optimization strategy Complexity in action: isobaric glycan separations Implications of Complexity on Analysis of Released Glycans
Protein N-linked Glycan Analysis Release of glycoprotein Asn-linked glycans by PNGase F is a well established approach, allow analysis of complex mixtures of glycans by a variety of methods. Various additional exoglycosidases and endoglycosidases can be helpful in structure elucidation. Considerable work has been accomplished for N-linked analysis of native glycans, as well as permethylated glycans using MS (ToF and ESI), and MS coupled to high resolution separation methods, particulary CE and LC. Glycoprofiling of N-linked glycans, using HILIC (NP) combined with fluorescence detection and/or MS is becoming a standard for biopharmaceutical glycoproteins, including antibodies.
Analysis of PNGase Released and Labeled N-Glycans Release of protein N-linked glycans using PNGase F releases oligosaccharides with a free reducing terminus (alditol) that is readily labeled by amines via formation of a Schiff s base, which can be reduced readily. Many amines have been applied to labeling glycans, (Harvey, 211, J. Chrom. 879). In the current work Procainamide is favored due to reported improvements in ESI-MS detection (Klapoetke, et al., 21, J. Pharm. Biomed. Anal. 53) Typical Labeling Conditions Glycan in water (up to 1% volume) 9+% volume of:.4 M procainamide 1M sodium cyanoborohydride in 3% acetic acid/7% DMSO 12-16 hr reaction at 37 C H 2 N SEC cleanup on Sephadex G-1 minicolumn Absorbance Detection 3 nm or Fluorescence Ex 33/Em 38 nm O N H Mass: glycan + 219.32 N
Superficially Porous Particles (Fused-Core ) Porous Shell SEM Particle Cross-section Solid Core 1.7 µm 2.7 µm.5 µm Low back pressure due to the particle design (solid core with a porous shell) Specialized high pressure HPLC equipment is optional Not necessary to filter samples and mobile phase since 2 µm frits are not as small as needed for sub-2-µm High resolution is maintained at high flow rates (flat C-term in van Deemter plot)
Silane Surface Modifications with Polar Functional Groups Support effective HILIC retention Exhibit desired kinetic advantages of SPP morphology Reduce unfavorable ionic/coloumbic interactions Hydroxylic Functional Groups FG Mono-OL O O H O H L k Di-OL O H H O R 1 Si R 2 Tri-OL R L O H H O R L = silanol reactive leaving R 1 = R 2 or R 1 = R 2, R 1 = R L Eg. -Me, -OMe, -Cl, -DiiPr H O Pentan-OL AKA Halo Penta-HILIC H O O H O H O H
Effect of Linear Velocity on Penta-HILIC Column Efficiency 4.6 mm ID x 5 mm; 9% AcN/1 mm NH 4 Form 3., 25 C; 1 ml, 5 ng Adenosine 2D Graph 2 6 Data fitted to Knox Equation 5 Reduced Plate Height 4 3 Silica HILIC 2 1 Penta-HILIC (minima 1.7;.9 ml/min) 1 2 3 4 5 6 7 8 9 Mobile Phase Velocity, mm/sec
Column Efficiency Comparisons Pam-G5 2.1 (2. mm) ID x 15 mm, 6 C, k 6, 5 mm Ammonium Formate Aqueous, ph 4.4.5 ul Injection (5 pmol), Abs. 3 nm Plate Height (µm) 4 Penta-HILIC, 72% AcN P = 147 bar 1.7 µm Amide, 69% AcN 3 3. µm Amide, 67% AcN P = 331 bar 2 P = 718 bar 1..5 1. Flow Rate
Penta-HILIC Separations of Labeled Oligosaccharides and Glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, 77.5-56.5% AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS (MS-22, (+) 4.2 kv, 4-2 with SIM) 6. mv Detector A 3nm 5.5 5. 4.5 4. 3.5 3. 2.5 2. 1.5 1..5..178.51.239.552.697 1.575 1.292 1.889 Pam-G2 3.138 5.141 7.893 11.154 14.614 18.3 21.22 24.158 26.879 -.5. 2.5 5. 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 3. 32.5 35. 37.5 4. 42.5 45. 47.5 5. 52.5 55. min 7 mv Detector A 3nm 6 5 4 3 2 1.136.196.551.737 1.19 1.489 1.314 1.75 1.636 9.136 Man 5 Man 6 12.427 15.635 16.4 18.36 17.445 18.576 Linear Dextran Standard Rs 11,1 = 4.3 Peak Capacity G22-G2 = 214 RNAase B N-linked Glycans Man 7 18.89 Man 8 21.87 21.427 Man 9 24.313 24.5 26.348 29.383 31.678 33.8 35.58 35.761 36.923 37.591 38.671 39.287 4.88 41.825 42.38 43.265 43.78 44.615 45.13 Pam-G22. 2.5 5. 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 3. 32.5 35. 37.5 4. 42.5 45. 47.5 5. 52.5 55. min 46.359 47.95 47.548 48.663 49.722 5.743 51.725 52.641 53.389 53.55 53.668 54.216 54.495 54.655 54.23
Penta-HILIC Retention of Labeled Oligosaccharides and Glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, 77.5-56.5% AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS (MS-22, (+) 4.2 kv, 4-2 with SIM) HILIC Retention of Glycans 6 Dextran RT (min) ManX Retention Time (min) 4 2 B T Fet Antenn 2 4 6 8 1 Hydroxyls
Penta-HILIC Separations of Abundant bov. a1-agp N-glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, 77.5-56.5% AcN (B) in 52.5 min., 6 C; 6 ml/min. MALDI of Permethylated Glycans % Intensity 1 9 8 7 6 5 4 3 2 1 (M-CH 2 +Na) + 2418.214 2448.2383 2435.2156 2462.2798 2793.729 2823.7673 2853.852 381.2273 399.243 3111.2485 3129.2595 3141.2791 3155.3137 3185.3384 3215.3547 3474.822 355.8582 3547.954 367.2 3714.2493 3759.354 4779.2 mv Detector A 3nm 12 11 1 9 8 7 6 5 4 3 2 1 2191. 2536.2 2881.4 Mass (m/z) 3226.6 3571.8 3917. HILIC of End-labeled Glycans.315.111.18.552.717 1.426 1.179 1.53 1.365 1.736 2.28 2.37 2.131 3.24 3.471 23.189 24.472 24.681 25.232 25.756 25.564 28.586 29.278 29.23 29.6 29.795 3.64 3.776 31.49 31.482 31.773 32.46 32.444. 2.5 5. 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 3. 32.5 35. 37.5 4. 42.5 45. 47.5 5. 52.5 55. min 33.424 33.85 34.453 34.777 35.333 35.785 36.57 36.682 37.584 36.98 38.484 37.823 38.723 39.66 39.879 4.513 41.293 42.99 42.95 43.71 47.511 54.78
HILIC Separations of a1-agp N-glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, 77.5-56.5% AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 15 mv Detector A 3nm 125 32.444 1 33.424 75 31.482 5 25 23.189 24.472 24.681 25.232 25.564 25.756 28.586 29.23 29.278 29.6 29.795 3.64 2. 21. 22. 23. 24. 25. 26. 27. 28. 29. 3. 31. 32. 33. 34. 35. 36. 37. min 3.776 31.49 31.773 32.46 33.85 34.453 34.777 35.333 35.785 36.57 36.682 36.98 7 6 5 2:176.5(+) : HexNAc 4 Hex 5 NeuAc 1 2:184.5(+) : HexNAc 4 Hex 5 NeuGc 1 2:1222.7(+) : HexNAc 4 Hex 5 NeuAc 2 2:123.(+) : HexNAc 4 Hex 5 NeuAc 1 NeuGc 1 2:1238.(+) : HexNAc 4 Hex 5 NeuGc 2 BNeuAc 2 BNeuAc 1 NeuGc 1 BNeuGc 2 4 BNeuAc BNeuGc 3 2 1 2. 21. 22. 23. 24. 25. 26. 27. 28. 29. 3. 31. 32. 33. 34. 35. 36. min
High Resolution HILIC Separations of asialo-fetuin N-glycans 2.1 mm ID x 3 mm; 5 mm Ammonium Formate, ph 4.4, 7-55% AcN (B) in 9min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 7 65 6 55 5 2:931.4(+): HexNAc 4 Hex 5 2:1114.1(+): HexNAc 5 Hex 6 2:1187.2(+): HexNAc 4 Hex 5 dhex 1 T 13.717 45 4 35 B 9.473 13.325 3 25 2? 15 1 5 8.834 9.23 13.349 13.731 14.75 15.229 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 min 9 mv Detector A 3nm 8 7 6 5 4 3 2 1 6.497 7.77 8.478 8.792 9.237 9.483 1.16 1.716 11.1 11.28 13.337 13.735 14.475 14.795 15.229 16.46 16.941 17.256 17.58 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 min 19.356 19.987 2.38 21.927 22.421 1. 7.5 5. 2.5 mv Detector A 3nm 8.478 8.792 8. 8.5 9. 9.5 min 9.237 9.483
High Resolution HILIC Separations of Fetuin N-glycans 2.1 mm ID x 3 mm; 5 mm Ammonium Formate, ph 4.4, 7-55% AcN (B) in 9min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 1. mv Detector A 3nm 7.5 5. 2.5 6.61 8.614 1.278 11.248 1.94 13.625 14.337 16.71 17.26 18.33 2.6 22.72 24.65 24.95 25.377 27.21 28.12 29.217 29.88 3.317 31.97 31.924 33.536 34.81 34.39 34.817 35.263 35.8 36.253 36.735 38.2. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. min 39.71 4.698 41.233 42.31 43.489 44.344 45.127 47.127 48.341 48.9 5.186 51.559 52.186 54.252 55.1 57.53 57.94 59.874 64.58 67.578 69.362 72.45 3 275 25 225 2:1551.(+): HexNAc 5 Hex 6 NeuAc 3 2:1696.6(+): HexNAc 5 Hex 6 NeuAc 4 2:1842.2(+)(1.): HexNAc 5 Hex 6 NeuAc 5 67.549 TNeuAc 5 2 175 42.31 15 45.15 125 1 75 5 TNeuAc 3 39.7 43.461 48.361 52.142 54.28 55.22 TNeuAc 4 57.945 25 1 2 3 4 5 6 7 8 9 min
High Resolution HILIC Separations of Fetuin N-glycans 2.1 mm ID x 3 mm; 5 mm Ammonium Formate, ph 4.4, 7-55% AcN (B) in 9min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) mv Detector A 3nm 7 6 25.377 28.12 42.31 45.127 55.1 57.94 5 4 3 2 1 6.61 8.614 1.278 1.94 11.248 13.625 14.337 16.71 17.26 18.33 2.6 22.72 24.65 24.95 27.21 29.217 29.88 3.317 31.97 31.924 33.536 34.81 34.39 34.817 35.263 35.8 36.253 36.735 38.2 39.71 4.698 41.233 43.489 44.344 47.127 48.341 48.9 5.186 51.559 52.186 54.252 57.53 59.874 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 3. 32.5 35. 37.5 4. 42.5 45. 47.5 5. 52.5 55. 57.5 min 15 125 2:931.4(+): HexNAc 4 Hex 5 2:17.(+): HexNAc 4 Hex 5 NeuAc 1 2:1222.7(+): HexNAc 4 Hex 5 NeuAc 2 25.383 27.986 BNeuAc 2 1 75 5 25 9.633 22.715 (ISD) 45.216 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. min
HILIC Separations of bov. a1-agp N-glycans (tri-sialated) 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, 77.5-56.5% AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 5 mv Detector A 3nm 45 4 33.424 35 3 36.98 25 2 36.57 37.823 15 1 5 33.85 34.453 34.777 35.333 35.785 36.682 37.584 38.484 38.723 39.66 39.879 4.513 41.293 42.99 42.95 33. 33.5 34. 34.5 35. 35.5 36. 36.5 37. 37.5 38. 38.5 39. 39.5 4. 4.5 41. 41.5 42. 42.5 min 5 45 4 35 2:1367.6(+): HexNAc 4 Hex 5 NeuAc 3 2:1375.6(+): HexNAc 4 Hex 5 NeuAc 2 NeuGc 1 2:1383.6(+): HexNAc 4 Hex 5 NeuAc 1 NeuGc 2 2:1391.6(+): HexNAc 4 Hex 5 NeuGc 3 BNeuAc 2 NeuGc 1 3 25 BNeuAc 3 BNeuAc 1 NeuGc 2 2 15 BNeuGc 3 1 5 33. 34. 35. 36. 37. 38. 39. 4. 41. 42. min
Conclusions A novel hydroxylated Fused-core silica HILIC packing material has high utility for end-labeled Glycan separations. The HILIC material and method described can be operated in high resolution mode, permitting analysis of complex mixtures of released N- linked protein glycan samples to be resolved, or in very high speed mode, to enable high throughput glycan analysis. High resolution HILIC resolves isobaric glycans, which are abundant (and complex). Variations in isobaric glycan profiles between proteins is very possible. Quantitation by end-labeling using UV or Fluorescence detection only could be problematic, detection by MS only will not easily reveal isomers.
Acknowledgements Penta-HILIC AMT Thank you for your Attention! Dr. J. DeStefano, Dr. J.J. Kirkland, Dr. S. Schuster NIH for Financial Support NIH NIGMS GM99355, GM93747 Shimadzu Corp early access to high sensitivity flow cell