Characterization and Quantification of Highly Sulfated Glycosaminoglycan Isomers by Gated-Trapped Ion Mobility Spectrometry Negative Electron Transfer Dissociation MS/MS

Abstract

Glycosaminoglycans (GAGs) play vital roles in many biological processes, and are naturally present as complex mixtures of polysaccharides with tremendous structural heterogeneity, including many structural isomers. Mass spectrometric analysis of GAG isomers, in particular highly sulfated heparin (Hep) and heparan sulfate (HS), is challenging because of their structural similarity and facile sulfo losses during analysis. Herein, we show that highly sulfated Hep/HS isomers may be resolved by gated-trapped ion mobility spectrometry (gated-TIMS) with negligible sulfo losses. Subsequent negative electron transfer dissociation (NETD) tandem mass spectrometry (MS/MS) analysis of TIMS-separated Hep/HS isomers generated extensive glycosidic and cross-ring fragments for confident isomer differentiation and structure elucidation. The high mobility resolution and preservation of labile sulfo modifications afforded by gated-TIMS MS analysis also allowed relative quantification of highly sulfated heparin isomers. These results show that the gated-TIMS-NETD MS/MS approach is useful for both qualitative and quantitative analysis of highly sulfated Hep/HS compounds in a manner not possible with other techniques.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides

“…IMMS separated standards display distinct characteristics. Since we and others have shown that IMMS can resolve HS saccharide isomers as large as octasaccharides 19,20,22,23 , we reasoned that it would have significant utility for HS sequencing in concert with the library of standard oligosaccharides. In IMMS molecular ions are transported through a gas-filled cell aided by a weak electric field, where they are separated according to their mass, charge, size and shape, which enables the differentiation of isomers.…”

“…GAG chain complexity is a result of heterogeneity from sulfation and epimerisation, and extended linear units. Preliminary results from our lab and others have shown that IMMS is a promising strategy for the gas phase separation of isomeric GAG oligosaccharide structures [18][19][20][21][22][23][24] .…”

Analysis of the Glycosaminoglycan Chains of Proteoglycans

“…22 Also, electron-based ion activation largely prevents the loss of sulfo group. 23 Although the loss of sulfo groups can also be prevented by deprotonation of sulfo groups or addition of metal adducts. 21 Moreover, the application of EDD or NETD can provide information of the same and different ions to aid the deconvolution of the oligosaccharide structure for GAG sequencing.…”

IR action spectroscopy of glycosaminoglycan oligosaccharides

“…In recent years, significant progress has been made in using novel electronbased ion activation methods (EDD [10], EID [11], NETD [12]) and ultraviolet photodissociation (UVPD) [13] to improve the capabilities of MS to overcome the unique challenges of GAG analysis. Another promising direction is to employ ion mobility spectrometry (IMS) to separate isomers or isomeric fragments prior to MS analysis [14,15]. A complementary method to MS-based approaches is Fourier transform infrared (FTIR) spectroscopy, which has been employed for the spectroscopic profiling of intact biological samples [16,17].…”