Enhanced protocol for quantitative N-linked glycomics analysis using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™

Autor: Erin S. Baker, Karen E Butler, Jaclyn Gowen Kalmar, David C. Muddiman
Rok vydání: 2020
Předmět:
Zdroj: Anal Bioanal Chem
ISSN: 1618-2650
1618-2642
DOI: 10.1007/s00216-020-02892-2
Popis: The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods has been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT ™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, and reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared to the standard method. Furthermore, the identification of low abundance glycans, such as (Fuc)(1)(Gal)(2)(GlcNAc)(4)(Man)(3)(NeuAc)(1) and (Gal)(3)(GlcNAc)(5)(Man)(3)(NeuAc)(3,) was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.
Databáze: OpenAIRE
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