Development and application of a sensitive phosphonium-hydrazide oligosaccharide labelling reagent in capillary...

Glycosylation is one of the most ubiquitous post-translational modifications (PTM) of proteins. Although the ionization efficiency of native glycans is fairly low, with the assistance of chemical derivation strategies, mass spectrometry (MS) has been extensively used in glycomics because of its high sensitivity, accuracy, and speed. In this study, a novel glycan labelling reagent, (4-hydrazidebutyl) triphenylphosphonium bromide (P₄HZD), with a permanent positive charge was developed. The comprehensive capabilities of P₄HZD for MS analysis of oligosaccharides were evaluated in detail using maltodextrin as a standard. This labelling reagent can be used in common biological MS techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. The MS signal intensity of maltodextrin species could be enhanced up to 96-fold in MALDI-MS by labelling with P₄HZD, making P₄HZD favorable for MALDI-MS-based high-throughput screening of oligosaccharides. Moreover, P₄HZD-labelled oligosaccharides with a degree of polymerization (DP) from 1 to 18 could be separated and analysed by capillary electrophoresis (CE) combined with positive ion mode ESI-MS. In comparison with a commercialized oligosaccharide tag, Girard's reagent P (GirP), P₄HZD was more effective for enhancing the signal of oligosaccharides in the middle or higher mass range using both ESI and MALDI ion sources. Two biologics, immunoglobulin G 2 (IgG 2) and fusion protein (FP), were chosen as model complex biological samples to test the efficacy of detection and separation of oligosaccharides by MALDI-MS and CE-ESI-MS analysis with P₄HZD labelling. The results indicated that P₄HZD is a promising labelling reagent for the detection of oligosaccharides in complex biological samples. The tandem workflow combines the strengths of MALDI-MS and CE-ESI-MS to fulfil the analytical demands of high-throughput screening, while affording good separation.