Enhanced protocol for quantitative N -linked glycomics analysis using Individuality Normalization when Labeling with Iso
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RESEARCH PAPER
Enhanced protocol for quantitative N-linked glycomics analysis using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ Jaclyn Gowen Kalmar 1 & Karen E. Butler 1 & Erin S. Baker 1,2 & David C. Muddiman 1,2,3 Received: 14 May 2020 / Revised: 9 August 2020 / Accepted: 17 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 have 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, 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 with 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. Keywords Glycomics . Orbitrap . Sample preparation . INLIGHT™ . N-linked glycans . HILIC . PGC . Reversed-phase . Chromatography
Introduction Glycosylation of proteins is a crucial post-translational modification associated with intra- and extracellular communicaElectronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02892-2) contains supplementary material, which is available to authorized users. * David C. Muddiman [email protected] 1
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA
2
Center for Human Health and the Environment, North Carolina State University, Raleigh, NC 27695, USA
3
Molecular Education, Technology, and Research Innovation Center (METRIC), North Carolina State University, Raleigh, NC 27695, USA
tion [1], protein folding and stability [2], and protein trafficking [3]. Since glycan properties, including protein binding locations, monomer linkages, and synthesis site and time, greatly dictate their functional roles, further investigations are greatly needed. N-linked glycan studies are of great interest for assessment of disease on
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