Integrated Pipeline of Isotopic Labeling and Selective Enriching for Quantitative Analysis of N-Glycome by Mass Spectrometry

Abstract

Quantitative N-glycomics can reveal abnormal expression of N-glycan in diseases. However, mass spectrometry (MS)-based N-glycome quantitative analysis is still technically challenging. To achieve the quantitation of Nglycome with high accuracy and sensitivity, it is required to efficiently label the N-glycans with isotopic tags and selectively enrich N-glycans to avoid suppression from other substances. Herein, we developed an integrated pipeline that combines isotopically fluorous tag labeling and fluorous solid-phase extraction to quantitatively analyze the N-glycome by MS. In this strategy, the N-glycans were labeled with light and heavy aminoxy-functionalized fluorous tags (PFBHA and PFBHAd 2 ) through the oxime click reaction. Then through the fluorous solid-phase extraction, the fluorous tag labeled N-glycan could be purified from contaminants like salts and proteins for the following quantitative analysis by mass spectrometry. This new approach enables selective purification (molar ratio of glycan to protein at 1:100) and accurate (R 2 > 0.99) and reproducible (coefficient of variation (CV)) < 25%, n = 6) quantitation of Nglycans within 2 orders of magnitude. Uniquely, diagnostic ions (D and [D-221]) were generated in tandem MS analysis after fluorous tags labeling, which could be used to deduce the composition of the 6-antenna and to distinguish isomers. Finally, this strategy was successfully applied to analyze the N-glycan changes in human serum associated with hepatocellular carcinoma (HCC). Fifteen N-glycan compositions with bisecting GlcNAc, sialic acid, and core fucosylation showed significant differences in HCC serum.

Heterogeneities of Site-Specific N-Glycosylation in HCC Tumors With Low and High AFP Concentrations

“…Unlike the previously reported studies of serum glycosylation where sialylated biantennary glycans were the main glycans, high mannose glycans also accounted for a large proportion of N-glycans in tissues. This is due to the fact that during the synthesis of N-glycans, high mannose is mainly in the Golgi and endoplasmic reticulum (intracellular), while complex glycans are mainly extracellular (36).…”

gQuant, an Automated Tool for Quantitative Glycomic Data Analysis

“…The accuracy and feasibility of gQuant was assessed by quantitating PFBHA datasets ( Yang et al, 2019 ), in which glycans from model glycoproteins were derivatized with PFBHA or PFBHA-2 deuterium. As shown in Supplementary Table 4 , glycan derivatives were almost always found in the top-ranked results, as the matched mass tolerances were in ascending order.…”

“…The GREDIL dataset used NaBH 4 to reduce PNGase F-released glycans and incorporated 18 O + deuterium to form a delta mass of 3.0 Da between samples, while the PFBHA dataset used o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) and PFBHA-2 deuterium for sample glycan derivatization. Detailed methods of the two datasets were well described in previous publications ( Cao et al, 2015 ; Yang et al, 2019 ). The datasets were adopted as part of the following search parameters.…”

Recent Advances in Analytical Approaches for Glycan and Glycopeptide Quantitation

“…Early reports of deuterium-based isotopic tags were provided by Bowman and Zaia, who first assessed multiple novel compounds for tetraplex labeling (43) and later applied them for glycan and glycosaminoglycan quantitation (44). Numerous other deuterium-based isotopic labeling strategies have been employed: derivatization with phenyl-methyl-pyrazole (PMP) has been employed to produce a one-pot dual-channel labeling strategy for matrix-assisted laser desorption/ionization (MALDI)-based quantitation of O-glycans (45,46), which was also adapted for in-gel labeling without significant sample loss (47); stabilization and quantitation of sialic-acid-containing glycans was promoted through a solid-phase p-toluidine labeling strategy (48); duplex stable isotope labeling (DuSIL) was developed to discriminate neutral and sialylated glycans without the need for synthesis (49)(50)(51); isomer-specific quantitation of sialic-acid-containing glycans was achieved through Glycoqueing, which enabled sialoglycan stabilization, isomer-specific elution order, and boosted MS signal (52); and quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) facilitated glycan release and labeling in a single step (53). Other methods for MS 1 level quantitation are reported by Yang et al, (54) who employed a metal chelating agent (p-NH 2 -Bn-DOTA) and rare earth metals to provide a 10 Da mass shift and near 100% labeling efficiency, and the quantification of N-glycan types presented by Li et al (55) that couples endoglycosidase digestion with channel labeling to provide an enrichment-friendly three-plex labeling strategy composition.…”