Procainamide labelling: A reliable and flexible approach to glycan analysis

Procainamide labelling permits glycan identification by either mass spectrometry or (U)HPLC, and because of its improved ionisation efficiency compared to 2AB labelling it can permit identification of minor glycans (<1% relative peak area) by ESI-MS.

Ludger's procainamide labelling system is suitable for N-glycans, O-glycans, GSL-glycans, heparin or any sugar with a reducing terminus. Also, once labelled with procainamide the glycans can be incubated with exoglycosidases.

As well as labelling kits we offer a clean up system for use after labelling and a range of procainamide labelled glycan standards (including an N-glycan library and glucose homopolymer ladder). Using products together (including LudgerZyme PNGaseF, LZ-rPNGaseF-kit), N-glycan sample preparation can be carried out in one day (for 96 samples)

Workflow

LudgerTag Procainamide for glycan profiling and identification:

ludger procainamide glycan labelling workflow

Application Note

Ludger Procainamide Labelling Advantages Application Note

Advantages of Ludger procainamide labelling vs rapid/instant tags

Choosing between labelling strategies for glycan analysis using liquid chromatography with fluorescence detection (LC-FLR) and mass spectrometry (MS) is a difficult task. Even though 2-aminobenzamide (2-AB) labelling has been a gold standard method for glycan profiling and characterisation using LC-FLR as an analytical platform, new contenders such as procainamide and rapid/instant tags have been in use recently due to their comparable fluorescence, increased MS sensitivity and short processing times (Keser T, et al. Front Chem. 2018;6:324).

However, it should be noted that procainamide labelling offers several advantages over rapid/instant tags. Our new application note outlines the features, benefits and advantages of Ludger's Procainamide labelling technology in comparison to gold standard labelling (2-AB) as well as other rapid/instant tags.

  Click for pdf version of application note

Comparison Table

The table below provides a quick summary of the features and benefits of Ludger's procainamide labelling technology in comparison to competitor offerings:

Ludger Procainamide Rapid/instant labelling*
Sensitive fluorescence detection for relative glycan quantitation by (U)HPLC ✓✓✓✓✓ ✓✓✓✓✓
Sensitive ESI-MS and MS/MS detection for glycan structural analysis ✓✓✓✓✓ ✓✓✓✓✓
Sample processing speed ✓✓✓ ✓✓✓✓✓
Can label purified glycans and standards? YES NO
Use for O-glycans? YES NO
Use for GSL glycans? YES NO
Similar glycan derivatisation chemistry to 2-AB industry standard? YES NO
Suitable for exoglycosidase sequencing? YES Fluorophore dependent**
* rapid/instant labelling technologies use N-hydroxysuccinimide (NHS) activated fluorophores that react with the glycosylamine form of the glycan
** aminoquinoline labels are not compatible with Bovine Kidney Fucosidase (BKF)

Product/Ordering Information:

Glycan Labelling with Procainamide:

Ludger Quantitative Sialic Acid Release and DMB Labeling Kit
  • LT-KPROC-24
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LT-KPROC-VP24
  • LudgerTag Procainamide Glycan Labelling Kit, 2PB (24 samples)

Each kit is suitable for labelling 24 samples (N- and O-glycans) and contains the following components:

  • Procainamide dye (LT-PROC-01)
  • 2PB reductant (2-picoline borane) (LT-PB-01) or sodium cyanoborohydride reductant (LT-CYANOB-05)
  • 30% acetic acid in DMSO (LT-ACETIC-DMSO-01)

Note: LT-KPROC-VP24 kit contains 2-picoline borane as reductant and LT-KPROC-24 kit contains sodium cyanoborohydride as reductant.

Ludger Quantitative Sialic Acid Release and DMB Labeling Kit
  • LT-KPROC-96
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (96 samples)

Each kit is suitable for labelling 96 samples (N- and O-glycans) and contains the following components:

  • Procainamide dye (LT-PROC-96)
  • Sodium cyanoborohydride reductant (LT-CYANOB-96)
  • 30% acetic acid in DMSO (LT-ACETIC-DMSO-96)

Note: 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'.

Companion Products:

N-glycan Analysis:

N-glycan Release

N-glycan Enrichment

  • LC-PBM-96
  • LudgerClean PBM 96 clean-up plate *optional clean up prior to procainamide labelling

Post labelling clean-up

  • LC-PROC-96
  • LudgerClean Procainamide post labelling clean-up plate

Procainamide labelled N-glycan standards

O-glycan Analysis:

O-glycan Release

  • LL-HYDRAZ-A2
  • LudgerLiberate Hydrazinolysis Release Kit (12 samples)

O-glycan Enrichment

  • LC-CEX-A6
  • LudgerClean CEX cartridges (pack of 6)

Post labelling clean-up

  • LC-S-A6
  • LudgerClean S cartridges (pack of 6)

Related Ludger Publications & Posters

Ludger publication - Complex N-glycan breakdown by gut Bacteroids

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.
Nature Microbiology. 2019 Jun 3.
doi: 10.1038/s41564-019-0466-x

Ludger publication - Engineering and stable production of recombinant IgE for cancer immunotherapy and AllergoOncology

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 Apr;141(4):1519-1523.e9.
doi: 10.1016/j.jaci.2017.12.986

Ludger publication - Analysis of Three Epoetin Alpha Products by LC and LC-MS

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

Ludger publication - Variation of Human Salivary O-Glycome

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

Ludger publication - Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans

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 RP, Badía Tortosa C, Fernandes DL, Spencer DI.
Analytical Biochemistry. 2015 Oct 1;486:38-40.
doi: 10.1016/j.ab.2015.06.006

Ludger poster

Investigating the role of insect vector glycosylation in African sleeping sickness transmission: Characterisation of procainamide-labelled tsetse fly saliva N-glycans

Kozak RP, Wongtrakul-Kish K, Williams C, Fernandes DL, Perally S, Rose C, Spencer DI, Acosta-Serrano A
Presented at Glyco23: 23rd International Symposium on Glycoconjugates
Split, Croatia, September 2015

Ludger poster

Procainamide labelling as part of a flexible glycoprofiling system for monitoring of Gal-a1-3Gal related Glycosylation Critical Quality Attributes (GCQAs) of monoclonal antibody (mAb) therapeutics throughout the product life cycle

Kozak RP, Royle L, Liew LP, Spencer DI, Fernandes DL
Presented at WCBP 2016: 20th Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products
Washington DC, United States, January 2016

Ludger poster

A QbD-compatible approach for reliable measurement of sialic acid O-acetylation as a potential Glycosylation Critical Quality Attribute (GCQA) of erythropoietin (EPO) therapeutics

Fernandes DL, Thomson R, Gardner R, Liew LP, Kozak RP, Royle L, Strohfeldt K, Osborn H, Spencer DI
Presented at WCBP 2016: 20th Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products
Washington DC, United States, January 2016

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