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Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT®) (Application Note 37)

The INLIGHT® glycan tagging kit, developed by the David Muddiman group in collaboration with synthetic chemist Daniel Comins at North Carolina State University,1,2 represents the latest in glycan-labeling technology for the relative quantification of N-linked glycans by mass spectrometry.

  • Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT®) (Application Note 37)
  • NIST interlaboratory study on glycosylation analysis of monoclonal antibodies: comparison of results from diverse analytical methods
INLIGHT® was developed by David Muddiman and colleagues at North Carolina State University. This kit is designed for the relative quantification of enzymatically cleaved N-glycans in binary samples (i.e., case and control) by MS. The kit contains the following:
13C6-labeled and an unlabeled biphenyl reagent (4-phenethylbenzohydrazide, phenyl 2-GPN) for hydrazide tagging of free glycans*
unlabeled maltoheptaose oligosaccharide (for quality control of extraction, derivatization, and LC-MS)
detailed instruction manual for performing the INLIGHT strategy (see data file and data report below for maltoheptaose and fetuin)
*The phenyl 2-GPN reagent offers significant advantages (e.g., rapid and efficient derivatization, enhanced hydrophobicity and ionization efficiency; see references below). This facilitates the relative quantification of typically polar glycans by reversed-phase LC-MS.


  INLIGHT® Glycan Tagging Kit

  Glycan Standards and Glycan Quantitation Kit

  Example INLIGHT Data Files click here to download zip file

 INLIGHT Data Report

  Stable Isotope Standards for Mass Spectrometry


Frequently Asked Questions 

How many reactions can be performed with the INLIGHT kit?

The kit contains five vials each of the heavy and light phenyl 2-GPN reagent (0.25 mg/vial) that is sufficient for approximately 125 reactions (25 reactions/vial). The number of reactions depends on the starting concentration of glycan and the sample type. The supplied user manual outlines the step-by-step instructions for relative quantitation of N-glycans in a glycoprotein (fetuin A) and an oligosaccharide (maltoheptaose). For best results, it is recommended that laboratories optimize the tagging reaction (re: glyco-substrate ratio) to minimize excess tag and maximize reaction efficiency.

What is the representative dataset that is provided for the INLIGHT kit?

The data files contain example MS data obtained after applying the INLIGHT strategy to fetuin A (glycoprotein) and maltoheptaose (oligosaccharide). The corresponding “INLIGHT Data Report” provides a summary of the glycans identified in these data files.

Can the INLIGHT kit be used for O-glycan analysis?

Yes. Through adherence to an optimized INLIGHT hydrazide tagging reaction, successful derivatization of O-linked glycans can be achieved. The details of the optimized reaction are described in PMID: 29279989, with application to two glycoprotein standards (human immunoglobulin A and bovine submaxillary mucin) demonstrated for utility.

What is the INLIGHT reaction mechanism for N- and O-glycans?

Once the N-glycans are enzymatically released by PNGase F, the hydrophobic phenyl 2-GPN reagent is introduced at the reducing sugar under mild, acidic conditions. Chemical methods are alternatively used to deglycosylate O-linked glycoproteins. This has been demonstrated to work with INLIGHT via hydrazinolysis, which leaves the O-glycans in non-reduced form enabling their derivatization.


Kalmar, J.G.; Garrard, K.P.; Muddiman, D.C. 2021. GlycoHunter: An open-source software for the detection and relative quantification of INLIGHT®-labeled N-linked glycans. J Proteome Res, 20(4), 1855-1863. PMID: 33417767

Kalmar, J.G.;  Butler, K.E; Baker, E.S.; et al. 2020. Enhanced protocol for quantitative N-linked glycomics analysis using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)®Anal Bioanal Chem, 412(27), 7569-7579. PMID: 32844281

De Leoz, M.L.A.; Duewer, D.L.; Fung, A.; et al. 2020. NIST interlaboratory study on glycosylation analysis of monoclonal antibodies: comparison of results from diverse analytical methods. Mol Cell Proteomics, 19(1), 11-30. PMID: 31591262

King, S.R.; Hecht, E.S.; Muddiman, D.C. 2018. Demonstration of hydrazide tagging for O-glycans and a central composite design of experiments optimization using the INLIGHT® reagent. Anal Bioanal Chem, 410(5), 1409-1415. PMID: 29279989

Mangrum, J.B.; Mehta, A.Y.; Alabbas, A.B.; Desai, U.R.; Hawkridge, A.M. 2017 Comparative analysis of INLIGHT®-labeled enzymatically depolymerized heparin by reverse-phase chromatography and high-performance mass spectrometry. Anal Bioanal Chem, 409(2), 499-509. PMID: 27888308

Loziuk, P.L.; Hecht, E.S.; Muddiman, D.C. 2017N-linked glycosite profiling and use of Skyline as a platform for characterization and relative quantification of glycans in differentiating xylem of Populus trichocarpaAnal Bioanal Chem, 409(2), 487-497. PMID: 27491298

Hecht, E.S.; McCord, J.P.; Muddiman, D.C. 2016. A quantitative glycomics and proteomics combined purification strategy. J Vis Exp, 109PMID: 27023253

Hecht, E.S.; Scholl, E.H.; Walker, S.H.; Taylor, A.D.; Cliby, W.A.; Motsinger-Reif, A.A.; Muddiman, D.C. 2015. Relative quantification and higher-order modeling of the plasma glycan cancer burden ratio in ovarian cancer case-control samples. J Proteome Res10, 4394-4401. PMID: 26347193

Hecht, E.S.; McCord, J.P.; Muddiman, D.C. 2015. Definitive screening design optimization of mass spectrometry parameters for sensitive comparison of filter and solid phase extraction purified, INLIGHT® plasma N-glycans. Anal Chem87(14), 7305-7312. PMID: 26086806
Walker, S.H.; Taylor, A.D.; Muddiman, D.C. 2013. Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A novel glycan-relative quantification strategy. J Am Soc Mass Spectrom, 24, 1376-1384. PMID: 23860851
Walker, S.H.; Lilley, L.M.; Enamorado, M.F.; Comins, D.L.; Muddiman, D.C. 2011. Hydrophobic derivatization of N-linked glycans for increased ion abundance in electrospray ionization mass spectrometry. J Am Soc Mass Spectrom, 22, 1309-1317. PMID: 21953184 
Andrew Percy, PhD

Andrew Percy, PhD

Senior Applications Chemist – Mass Spectrometry

Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry and the MS ‘Omics Product Manager at CIL. His responsibilities minimally involve providing technical support, overseeing product development, identifying new product market opportunities, assisting in the analysis of product-related applications, and writing/reviewing marketing literature.

Kevin Millis, PhD

Kevin Millis, PhD

Senior Scientist, Application Development Manager

Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and mass spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.


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