Stable-Isotope Labeled Hydrophobic Hydrazide Reagents for the Relative Quantification of N-Linked Glycans by Electrospray Ionization Mass Spectrometry

Abstract

This study presents the development of stable-isotope labeled hydrophobic, hydrazide reagents for the relative quantification of N-linked glycans. The P2GPN ‘light’ (12C) and ‘heavy’ (13C6) pair are used to differentially label two N-linked glycan samples. The samples are combined 1:1, separated using HILIC, and then mass differentiated and quantified using mass spectrometry. These reagents have several benefits: 1) impart hydrophobic character to the glycans affording an increase in electrospray ionization efficiency and MS detection; 2) indistinguishable chromatographic, MS, and MS/MS performance of the ‘light’ and ‘heavy’ reagents affording relative quantification; and 3) analytical variability is significantly reduced due to the two samples being mixed together after sample preparation. Obtaining these analytical benefits only requires ~4 hours of sample preparation time. It is shown that these reagents are capable of quantifying changes in glycosylation in simple mixtures, and the analytical variability of the reagents in pooled plasma samples is shown to be less than ±30%. Additionally, the incorporation of an internal standard allows one to account for the difference in systematic error between the two samples due to the samples being processed in parallel and not mixed until after derivatization.

Quantitative Liquid Chromatography-Mass Spectrometry-Multiple Reaction Monitoring (LC-MS-MRM) Analysis of Site-specific Glycoforms of Haptoglobin in Liver Disease

“…Elegant isotopic labeling of glycans in cell culture, similar to SILAC labeling of peptides, was also developed but remains to be used more widely (24,26). Methods for relative quantification of detached glycans based on the labeling of the reducing end or sialic acids represent further efforts to improve mass spectrometric quantification of detached N-glycans in this fast growing field of science (11,(27)(28)(29)(30).…”

Quantitative Glycomics Strategies

“…3D) (47). Two samples were labeled as light and heavy, respectively, prior to being mixed at a 1:1 ratio and undergoing LC/MS analysis.…”

“…The heavy and light N-glycans have identical chromatographic properties, MS results, and MS/MS performance. The reagents are capable of quantifying changes in the glycosylation of simple mixtures and pooled plasma samples in the presence of internal standards (47).…”

“…These hydrazides were systematically evaluated to determine which would effectively increase the relative abundance of N-linked glycans detected via ESI-MS. Phenyl2-GPN is a hydrazide that has a second phenyl ring, which increases the hydrophobicity and the ionization efficiency of the sample. The intensities of Phenyl2-GPN-labeled N-glycans derived from 50 l human plasma increased 10% relative to the same glycans labeled with other reagents possessing a single phenyl ring (47).…”

“…Five hydrazide reagents were recently synthesized: 2-phenylacethydrazide, 3-phenylpropane hydrazide, 4-phenylbutane hydrazide, 5-phenylpetane hydrazide, and 4-phenylbenzohydrazide (Phenyl2-GPN) (47). These hydrazides were systematically evaluated to determine which would effectively increase the relative abundance of N-linked glycans detected via ESI-MS. Phenyl2-GPN is a hydrazide that has a second phenyl ring, which increases the hydrophobicity and the ionization efficiency of the sample.…”