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

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 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 Nand 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™ Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. http://www.springer.com/gb/openaccess/authors-rights/aam-terms-v1

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

“…Recently, Muddiman and coworkers [151] reported a modified method of INLIGHT TM labeling which helped further to increase the ionization efficiency of labeled N ‐glycans and enhanced the sensitivity. By reducing the concentration of INLIGHT TM reagent and reaction time, lowering the reaction temperature, the signal intensity of N ‐glycans derived from model glycoprotein and biological samples has been significantly increased.…”

Utilizing liquid chromatography, ion mobility spectrometry, and mass spectrometry to assess INLIGHT™ derivatized N-linked glycans in biological samples

“…Ten to 50 μg of each glycan standard or 50 μg of maltoheptaose were placed in a vacuum concentrator until completely dry. The enzymatically released glycans, 50 μg maltoheptaose, and 10-50 μg of the N-linked glycan standards were derivatized with natural (NAT)-and stableisotope-labeled (SIL) INLIGHT™ reagents using a slight modification of a protocol that has been described previously [17]. The INLIGHT™ reagents were solubilized in 1 mL of LC-MS-grade methanol and vortexed for 10 min to ensure complete solubilization of the derivatization reagent, ensuring a final concentration of 1 mg/mL.…”

“…While maltoheptaose is not an N-linked glycan, this oligosaccharide is a reducing sugar and as a result is able to undergo the hydrazide chemistry necessary to affix the INLIGHT™ reagent [24]. Both the N-linked glycan standards and maltoheptaose were derivatized using a 1:1 mixture of the NAT and SIL reagents, then resuspended in MPA prior to analysis with nLC-IMS-MS [17]. As previously mentioned, glycans derivatized using the INLIGHT™ strategy display two distinctive isotopic distributions which is imparted by the use of both a NAT and 13 C 6 SIL label, facilitating rapid data analysis and relative quantitation of labeled glycans.…”

“…The hydrophilicity of glycans also complicates MS analyses when using an electrospray ionization (ESI) source, as ESI is inherently biased towards hydrophobic analytes [16,17]. To overcome this challenge, several types of derivatization chemistry have been employed to increase the overall hydrophobicity of N-linked glycans and improve their detection in both MS and spectroscopic methods.…”

“…The use of both a NAT and 13 C 6 incorporated SIL in INLIGHT TM enables the relative quantification of N-linked glycans in complex biological samples as the NAT and SIL INLIGHT™ tags have an exact mass difference of 6.0201 Da [25]. Additionally, the use of 13 C 6 prevents overlapping isotopic distributions, which may skew quantitative assessments [23][24][25] and allows for LC coelution of the NAT and SIL glycans [17,24,25,27]. Furthermore, this derivatization method does not require significant sample clean-up and has been demonstrated to work well within a filter-aided N-glycan sample (FANGS) preparation strategy [26].…”