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Abstract No.: TuOr-10
Speaker: I. Jonathan Amster
Session: Glycomics and Oligosaccharides
Presentation date: Tue, Aug 29, 2006
Presentation time: 11:50 – 12:10

Structure Studies of Sulfated Carbohydrates Using ECD and EDD

I. Jonathan Amster1, Jeremy J. Wolff1, Michael L. Easterling3, Lianli Chi2, Robert J. Linhardt2

1 University of Georgia, Athens, United States
2 Rensselaer Polytechnic Institute, Troy, United States
3 Bruker Daltonics, Billerica, United States

Correspondence address: I. Jonathan Amster, University of Georgia, Department of Chemistry, Cedar Street, Athens, Georgia, 30602-2556 United States.

Keywords: Carbohydrates, Structure Determination; Electron Capture; Electron Detachment; Fourier Transform ICR.

Novel aspect: First example of electron capture and electron detachment dissociation for a sulfated carbohydrate.


Glycosaminoglycans (GAGs) are complex, linear, and highly sulfated polysaccharides. The sulfated regions of GAGs bind numerous biological factors and play an important role in many biological processes such as blood coagulation, signal transduction, and pathway regulation. Structure elucidation of GAGs is difficult because of their large size, varying degrees of sulfation, and the instability of sulfate groups, particularly during MS/MS. Determination of the sites of sulfation on GAGs using MS/MS requires abundant cross ring fragmentation without loss of the labile SO3 group. We present here the results of electron capture dissociation (ECD) and electron detachment dissociation (EDD) of some model sulfated oligosaccharides to demonstrate the suitability of these methods for characterizing this challenging class of molecules.

Materials and Methods
Sucrose octasulfate (SOS), heparin tetrasaccharide (HepTet), and heparan sulfate (HS) were used as model sulfated oligosaccharides. All samples were dissolved in 50:50 methanol:H2O to a concentration of approximately 800 µM. Samples were analyzed with a 7T Bruker ApexQe FTMS fitted with an Apollo II ESI source. Electrons were generated using an indirectly heated hollow cathode. Multiply charged precursor ions were isolated in the external quadrupole or ICR cell before dissociation by ECD or EDD.

Preliminary Results
Chemical modification of sulfate and charge manipulation during ESI have been used by others to reduce sulfate decomposition during MS/MS. As an alternative to these methods, we have investigated the use of electron-aided dissociation of multiply-charge positive and negative ions as a means to generate fragmentation data that can identify the sites of sulfation in GAG oligosaccharides.

Initial studies were carried out on SOS with Na+ as the counter ion for the sulfate groups. The (M+2Na)2+ ion was used for ECD and the (M-2Na)2- ion was used for EDD. Both tandem MS techniques result in extensive fragmentation with minimal loss of SO3. HepTet was ionized with Na+ as the counter ion to make (M+2Na)2+ or (M-nNa)n-. ECD of the cationized species results in glycosidic bond cleavages and cross ring cleavages in the form of A and X ions. The effect of charge state on the dissociation products of HepTet during EDD has been investigated. The HS tetrasaccharide was analyzed by CAD and EDD. Due to the acidic nature of the molecule it was not possible to form doubly charged positive ions. EDD of the 2- ion results in glycosidic bond cleavage at each saccharide and extensive cross ring fragmentation of the reducing end sugars in the form of A ions. EDD of HS found no loss of the labile sulfate group. Thus ECD and EDD both show great potential for characterizing this challenging class of compounds.