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

Abstract

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~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™) 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™ 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™ derivatized glycans, facilitating rapid identification of putative glycans in complex biological samples.

A High-Throughput Ion Mobility Spectrometry–Mass Spectrometry Screening Method for Opioid Profiling

“…Three replicates of nine concentrations, ranging from 2 ng/mL to 1,000 ng/mL, were assessed for six of the opioids (buprenorphine, dihydrocodeine, fentanyl, norfentanyl, methadone, and oxycodone) using the SPE-IMS-MS platform. Both light and heavy forms of each opioid were used and the deuterated internal standards were readily observed within the data, regardless of matrix, as they are drift aligned with the analytes natural light isotope form as described previously and shown in Figure S4 . Peak areas for the comparisons were then determined using Skyline with IMS drift time filtering, and the resulting calibration curves for buprenorphine and oxycodone in water and urine are illustrated in Figure .…”

“…Both light and heavy forms of each opioid were used and the deuterated internal standards were readily observed within the data, regardless of matrix, as they are drift aligned with the analytes natural light isotope form as described previously and shown in Figure S4. 74 Peak areas for the comparisons were then determined using Skyline with IMS drift time filtering, and the resulting calibration curves for buprenorphine and oxycodone in water and urine are illustrated in Figure 5. Additional calibration curves are included in Figure S7.…”

Updates of the current strategies of labeling for N-glycan analysis

“…Permethylation is not discussed in the present review and this approach is described here [95]. More recently, a labeling strategy called Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT TM ) [96][97][98][99][100] has been developed using 4-phenethylbenzohydrazide (P2GPN) [101]. This strategy includes a reactive hydrazide group that allows for the rapid derivatization of released N-glycans without the need for reductive amination.…”

6-Plex mdSUGAR Isobaric-Labeling Guide Fingerprint Embedding for Glycomics Analysis

“…Through derivatization with chemical tags, functional groups are introduced into glycans which could facilitate ultraviolent/fluorescence spectrometric detection and/or boost MS signal responses . The most classic labeling reagent is 2-amino benzamide (2-AB), whereas various novel tags were developed recently, such as Rapifluor-MS and InstantPC, to reduce sample handling and improve labeling efficiency. − In addition, the utility of tandem MS for not only qualitative characterization but also quantitative analysis has gained popularity these days, − thereby several isobaric tags including QUANTITY, iART, and SUGAR , were customized to produce signature fragments during tandem MS (MS/MS) for relative quantification.…”