INLIGHT® Kit
Share
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
Resources
Glycan Standards and Glycan Quantitation Kit
Example INLIGHT Data Files click here to download zip file
Stable Isotope Standards for Mass Spectrometry
Frequently Asked Questions
How many reactions can be performed with the INLIGHT kit?
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.
References
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. 2017. N-linked glycosite profiling and use of Skyline as a platform for characterization and relative quantification of glycans in differentiating xylem of Populus trichocarpa. Anal 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, 109. PMID: 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 Res, 10, 4394-4401. PMID: 26347193

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.
More
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.
MoreRelated Products