Glycan Labelling System Sensitive profiling of glycan samples using - - PowerPoint PPT Presentation

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Glycan Labelling System Sensitive profiling of glycan samples using - - PowerPoint PPT Presentation

Mar 03, 2023 42 likes •278 views

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

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SLIDE 1

Sensitive profiling of glycan samples using (U)HPLC, ESI-MS, and LC-ESI-MS

LudgerT ag™ Procainamide Glycan Labelling System

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Procainamide Glycan Labelling |

In this presentation you will see how:

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Procainamide labelling is suitable for 25pmol – 25nmol quantity of sample and can identify minor glycan species This technology can be used for biopharma studies (e.g. IgG and EPO, N and O- glycosylation) and different biological samples (e.g. saliva and blood) The validation of the LudgerTag procainamide labeling system makes it suitable to GMP work

Procainamide

is applicable to both (U)HPLC, ESI-MS, and LC-ESI-MS analysis The incorporation of 2PB reductant makes the method easy/safer to use than sodium cyanoborohydride

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Procainamide Glycan Labelling |

Fluorescent labels and their suitability to (U)HPLC, MS

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5 stars = excellent, 4 stars = good, 3 stars = fair, 1 - 2 stars = poor

Fluorophore Structure (U)HPLC MS Comments Procainamide (PROC) ***** ***** Enhances MS ionization efficiency. Suitable for LC-MS systems, HPLC (HILIC, RP, WAX) and ESI- MS Low sample amount required Improves identification of very low abundance glycans 2-aminobenzamide (2-AB) **** **** The most widely used fluorescent tag. Suitable for HPLC (HILIC, RP, WAX) and MS Higher sample amount required for ESI-MS 2-aminobenzoic acid (2-AA) **** **** Suitable for HPLC (HILIC, RP) and MS Higher sample amount required for ESI-MS 2-aminopyridine (2-AP) *** **** Suitable for HPLC and MS Higher sample amount required for HPLC and MS analysis

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Procainamide Glycan Labelling |

The anatomy of procainamide

4-amino-N-(2-diethylaminoethyl) benzamide

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Fluorescent group for detection at Ex 310 and Em 370 nm in UHPLC 2-(diethylamino)ethyl group enhances the ionization efficiency in the positive ESI mode Site of attachment to glycan via reductive amination

N H O N H2N

Added mass 219.172 Da

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SLIDE 5

Procainamide Glycan Labelling |

LudgerTag Procainamide Labelling Kits

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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:

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’. Kit name Reductant Kit size LT-KPROC-24 Sodium cyanoborohydride 24 samples LT-KPROC-96 96 samples LT-KPROC-VP24 2-picoline borane 24 samples

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Procainamide Glycan Labelling |

Standards for procainamide workflow

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Process controls:

Glycoproteins – IgG, Fetuin Unlabelled glycans – including:

  • N- and O-glycans
  • Purified di-, tri-, tetraantennary glycans
  • High mannose glycans
  • Glycan libraries

For full list of standards check comparison table: https://www.ludger.com/system-suitability-standards

System suitability standards:

Labelled glycans:

  • Purified di-, tri-, tetraantennary glycans
  • High mannose glycans
  • Glycan libraries

Labelled Glucose Homopolymer (GHP)

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Procainamide Glycan Labelling |

LudgerTag Procainamide Workflow

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Released Glycans Procainamide Labelling Enrichment (Purification) and Clean up Ready for Analysis

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SLIDE 8

How we are using the LudgerT ag Procainamide Labelling System

  • for N-glycans
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SLIDE 9

Procainamide Glycan Labelling |

Workflow - LudgerTag Procainamide for N-glycan profiling and identification

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Glycoprotein

LZ-rPNGaseF-kit

PNGase F

LC-PBM-96

Binding Plate* *Optional clean-up, recommended for

  • ptimal results

LT-KPROC-VP24

  • r

LT-KPROC-24

  • r

LT-KPROC-96

Procainamide Labelling Kits

LC-PROC-96

LudgerClean PROC Plate

N-glycans Procainamide Labelled N-glycans Release Labelling Clean-up

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SLIDE 10

Procainamide Glycan Labelling |

Sample preparation within 1 day – N-glycans

LZ-rPNGaseF-kit

Glycan Release

LT-KPROC-VP24 LT-KPROC-24 LT-KPROC-96

Labelling

LC-PROC-96

Clean-up Analyse

Timing for processing of 96 samples

1 hr 1 hr Hands-on time Incubation time 1 hr 1 hr 2 hr

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Procainamide Glycan Labelling |

Typical Data

1454.70

1476.74 1855.38 1 2 3 4 6 x10 Intens. 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z

ESI-MS profile

Time

15.0 20.0 25.0 30.0 35.0 37.0

0.0e0 1.3e7 2.5e7 3.8e7 5.0e7 6.0e7

GU 6.00

Intensity

(U)HPLC profile

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SLIDE 12

Procainamide Glycan Labelling |

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

12 UPLC Chromatogram of 2-AB labeled glycans from mAb

Comparable in quantitation and in reliability, using a Waters ACQUITY UPLC Glycan BEH Amide Column (HILIC)

UPLC Chromatogram of Procainamide labelled glycans from mAb

0.00 10.00 20.00 30.00 40.00 50.00 60.00 1 2 3 4 5

Average Rel. % Area Peak number

2-AB Procainamide

Minutes 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00 260.00 280.00 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

1 2 3 4 5

Procainamide

Minutes

0.00

20.00 40.00 60.00 80.00 100.00 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

1 2 3 4 5

2-AB

500000 1000000 1500000 2000000 2500000 3000000 3500000

2-AB Procainamide Comparison of Signal Intensity (FLR) Comparison of Average Relative % Area

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Procainamide Glycan Labelling |

Procainamide Glycoprofiling by LC-MS

13 1 2 8 x10 Intens. [mAU] 2 4 6 8 x10 Intens. 16 18 20 22 24 26 28 30 32 Time [min]

Procainamide LC-FLR trace Procainamide ESI-MS trace

GU 5.66

  • Rel. % Area = 42.85%

2+ 1479.62 1+ 841.81 852.82 2+ 2 4 8 x10 Intens. 400 600 800 1000 1200 1400 1600 1800 m/z 441.24 587.31 740.81 991.34 1114.47 1276.56 1479.63 146.07 790.37 0.0 0.5 1.0 1.5 5 x10 Intens. 200 400 600 800 1000 1200 1400 m/z

MS spectra MS/MS spectra Glycan composition: H3N4F1 Possible structure: FA2

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Procainamide Glycan Labelling | ESI-MS and MS/MD data for HILIC peak 19 HILIC-(U)HPLC data for procainamide labelled human IgG -13 replicates

17.10 18.75 20.00 21.25 22.50 23.75 25.00 26.25 27.50 28.75 30.00 31.25 32.50 33.75 35.09

Time

Relative Fluorescence 1 2 3 4 5 6 9 7 8 10 11 12 13 14 15 16 17 18 19 20 21 22

  • Repeatable and robust
  • One system for relative

quantitation and identification of glycans (HILIC-(U)HPLC fitted with online MS)

Hex (H) HexNAc (N) Fuc (F) Neu5Ac (S) [M/Z]+ [M/Z]2+ [M/Z]3+ [M/Z]+ [M/Z]2+ [M/Z]3+ 2136.01 (H4N4F1S1-PROC) 1770.38 (H3N3F1S1-PROC) 1479.73 (H3N3F1-PROC) 790.40 (N2F1-PROC) 1989.88 (H4N4S1-PROC) 1712.63 (H5N3S1) 1317.74 (H2N3F1-PROC) 657.23 (H1N1S1) 1974.88 (H3N4F1S1-PROC) 1682.84 (H3N4F1-PROC) 1277.69 (H3N2F1-PROC) 587.38 (N1F1-PROC) 1932.84 (H4N3F1S1-PROC) 1642.77 (H4N3F1-PROC) 1114.59 (H2N2F1-PROC) 441.23 (N1-PROC) 1844.87 (H4N4F1-PROC) 1536.57 (H3N4-PROC) 819.29 (H2N1S1) 366.12 (H1N1) 864.34 nd 1251.11 834.41 Peak no 19 5 5 1 1 2501.01 1251.01 Calculated Registered Composition [M/Z] characteristic fragment ions (composition)

Average STDev CV Rep1 Rep2 Rep3 Rep4 Rep5 Rep6 Rep7 Rep8 Rep9 Rep10 Rep11 Rep12 Rep13 1 5.26 0.59 0.51 0.50 0.57 0.54 0.55 0.49 0.55 0.56 0.58 0.57 0.52 0.54 0.54 0.03 5.74 2 5.65 18.49 17.99 18.46 17.74 17.65 17.75 17.38 17.80 17.61 17.69 17.42 17.65 17.80 17.80 0.34 1.90 3 5.99 4.12 4.03 4.10 3.94 3.92 3.92 3.90 3.95 3.90 3.95 3.80 3.89 3.95 3.95 0.09 2.20 4 6.05 0.93 0.89 0.92 0.92 0.92 0.89 0.91 0.93 0.90 0.91 0.91 0.93 0.91 0.91 0.01 1.51 5 6.16 0.47 0.41 0.42 0.43 0.48 0.42 0.42 0.48 0.41 0.44 0.47 0.39 0.44 0.44 0.03 6.76 6 6.33 0.49 0.49 0.50 0.49 0.49 0.45 0.47 0.49 0.49 0.46 0.48 0.49 0.48 0.48 0.01 2.95 7 6.43 18.76 18.51 18.81 18.08 18.22 18.26 17.91 18.22 18.15 18.05 18.06 18.27 18.28 18.28 0.27 1.47 8 6.55 8.11 7.95 8.13 7.85 7.87 7.90 7.80 7.88 7.88 7.87 7.86 7.94 7.92 7.92 0.10 1.23 9 6.68 5.01 4.92 4.99 4.84 4.89 4.85 4.81 4.85 4.82 4.84 4.82 4.86 4.88 4.88 0.06 1.30 10 6.8 0.91 0.91 0.89 0.95 0.90 0.91 0.93 0.93 0.88 0.93 0.85 0.89 0.91 0.91 0.03 2.86 11 6.96 0.95 0.89 0.97 0.95 0.98 0.92 0.97 0.91 0.94 0.97 0.98 0.92 0.95 0.95 0.03 3.04 12 7.13 0.81 0.77 0.73 0.78 0.76 0.73 0.76 0.73 0.75 0.77 0.72 0.78 0.76 0.76 0.03 3.37 13 7.33 13.89 13.92 13.97 13.74 13.97 14.11 13.89 13.94 14.05 13.93 14.11 14.13 13.97 13.97 0.11 0.78 14 7.54 4.28 4.30 4.25 4.26 4.34 4.31 4.32 4.30 4.30 4.32 4.31 4.32 4.30 4.30 0.02 0.58 15 7.93 2.31 2.58 2.33 2.62 2.43 2.38 2.88 2.61 2.60 2.61 2.55 2.53 2.54 2.54 0.15 5.93 16 8.19 0.88 0.96 0.91 0.97 0.97 0.91 1.03 0.96 0.95 0.97 0.94 0.93 0.95 0.95 0.04 3.89 17 8.3 9.01 9.36 9.13 9.21 9.54 9.68 9.56 9.54 9.70 9.45 9.80 9.66 9.47 9.47 0.24 2.49 18 8.52 2.47 2.68 2.50 2.61 2.72 2.74 2.66 2.62 2.70 2.62 2.69 2.63 2.64 2.64 0.08 2.99 19 8.92 1.42 1.34 1.25 1.39 1.37 1.34 1.49 1.39 1.39 1.38 1.38 1.35 1.37 1.37 0.05 3.91 20 9.07 0.51 0.56 0.52 0.77 0.62 0.56 0.69 0.59 0.56 0.60 0.60 0.58 0.60 0.60 0.07 11.55 21 9.28 2.76 2.99 2.83 3.05 3.15 3.19 3.29 3.10 3.22 3.18 3.27 3.13 3.10 3.10 0.16 5.11 22 9.39 2.83 3.04 2.87 3.21 3.27 3.23 3.42 3.23 3.25 3.24 3.39 3.21 3.18 3.18 0.17 5.47 % Area % Area GU Peak Id

ESI-MS of peak no 19. ESI-MS/MS of peak no 19.

LC-MS analysis of human IgG

14 HILIC-(U)HPLC chromatogram of procainamide labelled human IgG -13 replicates

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Procainamide Glycan Labelling |

ESI-MS of selected (GU 8.47) procainamide labelled peak HILIC-(U)HPLC chromatograms of procainamide labelled N-glycans released from EPO (3 different manufacturers) LC-MS LC-MS ESI-MS/MS of selected (GU 8.47) procainamide labelled peak

LC-MS analysis of biosimilars

erythropoeitin, EPO

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Procainamide Glycan Labelling |

ESI-MS/MS of selected peaks chromatograms for procainamide labelled N-glycans released from tsetse fly saliva LC-MS LC-MS

Sleeping sickness occurs in 36 sub-Saharan Africa countries where there are tsetse flies that transmit the

  • disease. It is transmitted through the saliva of the tsetse

fly during feeding Salivary glycoproteins have been reported to:

  • facilitate host infection through binding and transport
  • f vector-borne diseases to host tissues
  • participate in host responses such as inflammation

and immune response

325.06 368.14 441.24 487.13 571.09 644.36 690.28 806.38 895.40 968.46 1057.37 1130.50 325.10 368.06 403.09 441.25 487.22 514.01 549.34 587.34 644.38 749.30 806.39 895.31 968.50 1005.56 1057.39 1096.60 1130.51 1203.42 1276.52 325.02 368.06 441.21 487.15 528.20 571.18 644.30 672.31 733.32 806.39 895.40 937.35 968.39 1057.47 1098.47 1130.51 1171.58 1260.46 1333.62 0.0 0.2 0.4 0.6 0.8 1.0 7 x10 Intens. 0.0 0.5 1.0 1.5 5 x10 2 4 6 8 5 x10 200 400 600 800 1000 1200 m/z 952.48 1114.53

a) b) c)

(U)HPLC profile of procainamide labelled N-glycans released from tsetse fly saliva

LC-MS analysis of complex biological samples

Tsetse fly saliva

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SLIDE 17

How we are using the LudgerT ag Procainamide Labelling System

  • for O-glycans
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Procainamide Glycan Labelling |

Workflow - LudgerTag Procainamide for O-glycan profiling and identification

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Glycoprotein O-glycans Procainamide Labelled O-glycans

LL-HYDRAZ-A2

Hydrazine release

LC-CEX-A6

CEX cartridge

LT-KPROC-VP24

  • r

LT-KPROC-24

  • r

LT-KPROC-96

Procainamide Labelling Kits

LC-S-A6

LudgerClean S cartridges

Release Labelling Clean-up Clean-up

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SLIDE 19

Procainamide Glycan Labelling |

Comparison of 2-AB and Procainamide Labelled O-glycans

19 UHPLC Chromatogram of 2-AB labelled O-glycans from bovine fetuin

Comparable in quantitation and in reliability, using a Waters ACQUITY UPLC Glycan BEH Amide Column (HILIC)

UHPLC Chromatogram of Procainamide labelled O-glycans from bovine fetuin Base Peak Chromatogram of 2-AB labelled O-glycans from bovine fetuin Base Peak Chromatogram of Procainamide labelled O-glycans from bovine fetuin

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SLIDE 20

Procainamide Glycan Labelling |

LC-ESI-MS/MS analysis of Procainamide Labelled O-glycans

20 UHPLC Chromatogram of Procainamide labelled O-glycans from bovine fetuin Base Peak Chromatogram of Procainamide labelled O-glycans from bovine fetuin

GU 4.91

  • Rel. % Area = 2.22%

Glycan composition: H2N2S2 Possible structure: di-sialylated core 2 O-glycan

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SLIDE 21

Procainamide Glycan Labelling |

HILIC-LC-ESI-MS of procainamide labelled O-glycans released from human saliva

LC-MS analysis of complex biological samples

Human saliva

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LC-MS

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SLIDE 22

Procainamide Glycan Labelling |

Related publications

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Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization-mass spectrometry

Kozak R, Tortosa C, Fernandes D, Spencer D. Analytical Biochemistry. 2014 October;486:38-40. doi:10.1016/j.ab.2015.06.006. Epub 1 October 2015

Variation of Human Salivary O-Glycome

Kozak RP, Urbanowicz PA, Punyadeera C, Reiding KR, Jansen BC, Royle L, Spencer DI, Fernandes DL, Wuhrer M. PLoS One. 2016 Sep 9;11(9):e0162824. doi: 10.1371/journal.pone.0162824. eCollection 2016.

Engineering and stable production of recombinant IgE for cancer immunotherapy and AllergoOncology

Crescioli S, Chiaruttini G, Mele S, Ilieva KM, Pellizzari G, Spencer DIR, Gardner RA, Lacy KE, Spicer JF, Tutt ANJ, Wagner GK, Karagiannis SN. Journal of Allergy and Clinical Immunology. 2018 Jan 31. pii: S0091-6749(18)30081-2. doi: 10.1016/j.jaci.2017.12.986. [Epub ahead of print]

Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content

Thomson RI, Gardner RA, Strohfeldt K, Fernandes DL, Stafford GP, Spencer DIR, Osborn HMI. Analytical Chemistry. 2017 Jun 20;89(12):6455-6462. doi: 10.1021/acs.analchem.7b00353. Epub 2017 Jun 9.

Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci

Briliūtė J, Urbanowicz PA, Luis AS, Baslé A, Paterson N, Rebello O, Hendel J, Ndeh DA, Lowe EC, Martens EC, Spencer DIR, Bolam DN, Crouch LI. Nat Microbiol. 2019 Jun 3. doi: 10.1038/s41564-019-0466-x. [Epub ahead of print]

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Procainamide Glycan Labelling |

CLICK to contact Rad

Dr Radoslaw Kozak Head of Glycoprofiling

rad.kozak@ludger.com

If you have a question

CLICK to contact Sales

Request a quotation

Sales Team

info@ludger.com

How to start using the Procainamide Glycan Labelling System

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