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

Glycosylation is a ubiquitous co- and post-translational modification involved in the sorting, folding, and trafficking of proteins in biological systems; in humans, >50% of gene products are glycosylated with the cellular machinery of glycosylation compromising similar to 2% of the genome. Perturbations in glycosylation have been implicated in a variety of diseases including neurodegenerative diseases and certain types of cancer. However, understanding the relationship between a glycan and its biological role is often difficult due to the numerous glycan isomers that exist. To address this challenge, nanoflow liquid chromatography, ion mobility spectrometry, and mass spectrometry (nLC-IMS-MS) were combined with the Individuality Normalization when Labeling with the Isotopic Glycan Hydrazide Tags (INLIGHT (TM)) strategy to study a series of glycan standards and those enzymatically released from the glycoproteins horseradish peroxidase, fetuin, and pooled human plasma. The combination of IMS and the natural (NAT) and stable-isotope label (SIL) in the INLIGHT (TM) strategy provided additional confidence for each glycan identification due to the mobility aligned NAT- and SIL-labeled glycans and further capabilities for isomer examinations. Additionally, molecular trend lines based on the IMS and MS dimensions were investigated for the INLIGHT (TM) derivatized glycans, facilitating rapid identification of putative glycans in complex biological samples.

Automated summary

Glycosylation is a common protein modification associated with various diseases, causing challenges in understanding the relationship between glycans and biological functions. The combination of IMS and MS technologies with the INLIGHT(TM) strategy enhances glycan identification confidence and accelerates the identification process in complex biological samples.