INLIGHT® Kit

Glycans participate in a large number of cellular, molecular, and biological processes and are implicated in a number of diseases (e.g., Alzheimer’s, cancer). To aid the identification and relative quantification of glycans by LC-MS, CIL offers the innovative INLIGHT® (Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tag) glycan-tagging kit. This kit employs both natural (NAT) and stable isotope-labeled (SIL; 13C6) phenyl 2-GPN reagents in the hydrazide derivatization of free glycans. While this tagging strategy was developed for N-glycans, it has also been adapted to O-glycan and heparin oligomer profiles. The INLIGHT kit contains five vials of NAT reagent and five vials of SIL reagent, which in total provides sufficient tagging for approximately 125 relative quantification experiments. The user manual provides step-by-step instructions for executing the INLIGHT strategy using maltoheptaose and fetuin A as examples. Data processing and analysis of derivatized glycans can be facilitated in GlycoHunter or Skyline.

Related Resources

➤ Stable Isotope Standards for Mass Spectrometry
➤ Stable Isotope-Labeled Mixtures, Sets, and Kits
➤ Mixtures, Sets, and Kits for MS ‘Omics and MS/MS Screening
➤ Glycan Standards and Glycan Quantitation Kit

Application Note
➤ Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™): A Novel N-Linked Glycan Relative Quantification Strategy

INLIGHT® Glycan Tagging Kit

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Isotope Label

Isotopic Enrichment Range

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Product Form 2

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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 nonreduced form enabling their derivatization.

Example References

Butler, K.E.; Kalmar, J.G.; Muddiman, D.C.; et al. 2022. Utilizing liquid chromatography, ion mobility spectrometry, and mass spectrometry to assess INLIGHT® derivatized N-linked glycans in biological samples. Anal Bioanal Chem, 414(1), 623-637. PMID: 34347113
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.; et al. 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, 53735-53752. PMID: 27023253
Hecht, E.S.; Scholl, E.H.; Walker, S.H.; et al. 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
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 Chem, 87(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.; et al. 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