Hydrophobic Derivatization of N-linked Glycans for Increased Ion Abundance in Electrospray Ionization Mass Spectrometry

Abstract

A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in order to increase the ion abundance of N-linked glycans in electrospray ionization mass spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion abundance, a property that can be further exploited in the analysis of glycans. The results of this study will direct the future synthesis of hydrophobic reagents for glycan analysis using the correlation between hydrophobicity and theoretical non-polar surface area calculation to facilitate the development of an optimum tag for glycan derivatization. The compatibility and advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly more abundant, and the detection of negatively charged sialylated glycans is enhanced.

Mass spectrometry of glycans

“…This property has been used to improve ionization, to add a stable isotope tag or to attach a chromophore for detection by High-performance Liquid Chromatography (HPLC) [28, 29]. Reductive amination works for N -linked glycans released enzymatically from proteins and for any glycan that contains a reducing end.…”

Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS

“…This signal enhancement is attributed to the increase in hydrophobicity of the tagged molecules. Previous work has shown that improved ESI-MS signal of analytes as a result of increased hydrophobicity is likely due to localization at the periphery of the electrospray droplet 9,16 . Not only was signal enhancement achieved for acidic carbonyls but more substantially for the neutral glucose, an aldehyde sugar which showed no appreciable signal when untagged at micromolar concentrations tested.…”

Hydrazino-s-triazine based labelling reagents for highly sensitive glycan analysis via liquid chromatography–electrospray mass spectrometry

“…The labelling reaction was carried out according to a previous method [35]. Ten labelling reagent solutions (50 μM) were prepared in MeOH/acetic acid (75/25, v/v) individually.…”

“…The labelling procedures were carried out referring to a literature with high labelling efficiency and minimized loss of sialic acid and fucose [35]. Fast and mild labelling process could be operated in nonreductive mode without the use of any toxic reagents.…”