Ultraviolet photodissociation of fondaparinux generates signature antithrombin-like 3-O-sulfated -GlcNS3S6S- monosacchar
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RESEARCH PAPER
Ultraviolet photodissociation of fondaparinux generates signature antithrombin-like 3-O-sulfated -GlcNS3S6S- monosaccharide fragment (Y3/C3) Adam M. Hawkridge 1,2 & Sven Hackbusch 3 Received: 23 May 2020 / Revised: 27 July 2020 / Accepted: 28 August 2020 / Published online: 18 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract The 3-O sulfate-modified -GlcNS3S6S- monosaccharide in heparin and heparan sulfate glycosaminoglycans (HSGAGs) is a relatively rare yet important modification that facilitates HSGAG-antithrombin binding and subsequent anticoagulant activity. Detecting this modification in complex HSGAG mixtures is a longstanding goal to identify novel 3-O-sulfated HSGAG-protein interactions with biologically significant functions. Tandem mass spectrometry has been applied to HSGAG structural analysis but is limited by the fact that traditional collision-induced dissociation techniques (e.g., CID, HCD) results in extensive sulfate loss prior to generating structurally informative glycosidic and cross-ring fragments. In the present study, we investigated the potential of ultraviolet photodissociation (UVPD) to generate structurally informative fragments from the synthetic heparin mimetic, fondaparinux, under electrospray conditions commensurate with hydrophilic interaction liquid chromatography (HILIC). The two predominant un-adducted precursors, [Fonda-2H+]2− and [Fonda-3H+]3−, were subjected to UVPD, CID, and HCD on an Orbitrap Fusion Lumos Tribrid mass spectrometer and the resulting fragmentation spectra directly compared. Close inspection of the UVPD data identified a unique peak at m/z 417.9425 that matched the Y3/C3 double glycosidic fragment of fondaparinux (i.e., -GlcNS3S6S-). Importantly, the 3-O-sulfated Y3/C3 fragment was generated predominantly from UVPD of the [Fonda-2H+]2− precursor, increased with activation time, and was observable using data-dependent HILIC-MS/MS UVPD analysis of fondaparinux spiked into a semi-complex HSGAG mixture. The discovery of this antithrombin-like 3-O-sulfated fragment provides a potential strategy for screening complex HSGAG mixtures in a data-dependent or data-independent acquisition mode to determine the presence of this therapeutic and biologically significant HSGAG modification.
Keywords Heparin . Mass spectrometry . 3-O Sulfation . UVPD . Glycosaminoglycans
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02925-w) contains supplementary material, which is available to authorized users. * Adam M. Hawkridge [email protected] 1
Department of Pharmaceutics and Pharmacotherapy & Outcomes Science, Virginia Commonwealth University School of Pharmacy, Richmond, VA 23298, USA
2
Institute for Structural Biology, Drug Discovery, and Development, Virginia Commonwealth University, Richmond, VA 23298, USA
3
Thermo Fisher Scientific, San Jose, CA 95134, USA
Heparin and heparan sulfate are closely related glycosaminoglycans (HSGAGs) that play significa
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