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.

No 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.

The analysis of N-linked glycans by mass spectrometry (MS) has been characterized by low signal-to-noise ratios and high limits of detection due to their hydrophilicity and lack of basic sites able to be protonated. As a result, every step in glycan sample preparation must be thoroughly optimized in order to minimize sample loss, contamination, and analytical variability. Importantly, properties of glycans and their derivatized counterparts must be thoroughly studied in order to exploit certain characteristics for enhancing MS analysis. Herein, the effectiveness of the incorporation of a permanent charge is studied and determined to hamper glycan analysis. Also, a procedure for glycan hydrazone formation is optimized and outlined where a large number of variables were simultaneously analyzed using a fractional factorial design (FFD) in order to determine which conditions affected the reaction efficiency of the hydrazone formation reaction. Finally, the hydrophobic tagging of glycans is shown to be a viable opportunity to further increase the ion abundance of glycans in MS.

The quantitative and confirmative performance of two different mass spectrometry (MS) techniques (high-resolution MS and tandem MS) was critically compared. Evaluated was a new extraction and clean-up protocol which was developed to cover more than 100 different veterinary drugs at trace levels in a number of animal tissues and honey matrices. Both detection techniques, high-resolution mass spectrometry (HRMS) (single-stage Orbitrap instrument operated at 50 000 full width at half maximum) and tandem mass spectrometry (MS/MS) (quadrupole technology) were used to validate the method according to the EU Commission Decision 2002/657/EEC. Equal or even a slightly better quantitative performance was observed for the HRMS-based approach. Sensitivity is higher for unit mass resolution MS/MS if only a subset of the 100 compounds has to be monitored. Confirmation of suspected positive findings can be done by evaluating the intensity ratio between different MS/MS transitions, or by accurate mass based product ion traces (no precursor selection applied). MS/MS relies on compound-specific optimized transitions; hence the second, confirmatory transition generally shows relatively high ion abundance (fragmentation efficacy). This is often not the case in single-stage HRMS, since a generic (not compound-optimized) collision energy is applied. Hence, confirmation of analytes present at low levels is superior when performed by MS/MS. Slightly better precision, but poorer accuracy (fortified matrix extracts versus pure standard solution) of ion ratios were observed when comparing data obtained by HRMS versus MS/MS.