Distinguishing Glucuronic from Iduronic Acid in Glycosaminoglycan Tetrasaccharides by Using Electron Detachment Dissociation

Abstract

Distinguishing the epimers iduronic acid (IdoA) versus glucuronic acid (GlcA) has been a longstanding challenge for the mass spectrometry analysis of glycosaminoglycan oligogosaccharides. In this work, electron detachment dissociation (EDD) and Fourier transform ion cyclotron resonance mass spectrometry is shown to provide mass spectral features that can distinguish GlcA from IdoA in heparan sulfate (HS) tetrasaccharides. EDD of HS tetrasaccharide dianions produces a radical species that fragments to produce information-rich glycosidic and crossring product ions which can be used to determine the sites of acetylation/sulfation. More significantly, EDD of HS tetrasaccharide epimers produces diagnostic product ions that can be used to distinguish IdoA from GlcA. These diagnostic product ions are not observed in the tandem mass spectra obtained by collisionally activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD) of the tetrasaccharides, suggesting a radical-initiated mechanism for their formation. Differences in the observed product ions obtained by EDD of the tetrasaccharide epimers can be rationalized by simple α-cleavage of an oxy-radical located at C2 or C3 or a radical at C3 or C4. These radicals are proposed to arise from a hydrogen rearrangement in which a hydrogen atom is transferred from the C2 or C3 hydroxyl group or C3 or C4 to a carboxy radical at C5. This hydrogen transfer depends on the proximity of the carboxy radical to the hydroxyl group on C2 or C3 or the hydrogen on C3 or C4, and is thus influenced by C5 stereochemistry. These epimer-sensitive fragmentations should allow this approach to be applied to the structural analysis of a wide variety of GAG oligosaccharides.

Electron detachment dissociation of neutral and sialylated oligosaccharides

“…Hydrogen addition and abstraction following ECD of peptides have been well documented [60,59], and have also been reported for EDD of peptides [45]. Following EDD of GAGs, hydrogen abstraction from Band C-type ions has also been noted, and observed as B Ϫ H, C Ϫ H, and C Ϫ 2H type ions [32,33]. Of particular interest is the product ion type C Ϫ 2H, which also has been reported by several investigators following positive-ion mode heCAD [58,[61][62][63][64] and which is observed following EDD of sialylated oligosaccharides in our examination.…”

“…During positive-ion mode heCAD, the precise mechanism that generates these ions is somewhat unclear [58]. In EDD, Wolff et al have proposed that electron detachment at a carboxylate group is followed by hydrogen transfer to form either an oxy radical at C-2 or a radical at C-3, which can lead to the formation of C Ϫ 2H product ions by ␣-cleavage ␣ [33]. Following EDD of GAGs, the complementary Z ϩ 2H ions (or Y Ϫ 16 Da species) were not observed.…”

“…Although most modern mass analyzers use lowenergy dissociation techniques such as low-energy CAD, several alternative fragmentation techniques have been developed and applied to oligosaccharides, including high-energy CAD [25][26][27], IRMPD [28,29], electron capture dissociation (ECD) [30,31], electron detachment dissociation (EDD) [32,33], and 157-nm photodissociation [34]. In particular, the use of ionelectron reactions for biomolecular structural characterization is rapidly expanding.…”

“…In EDD, polyanions are irradiated with Ͼ10 eV electrons, resulting in electron detachment and subsequent product ions. EDD has been applied to peptides [45][46][47], oligonucleotides [48,49], gangliosides [50], and recently glycosaminoglycans (GAGs) [32,33]. GAGs are linear polysaccharides consisting of repeating disaccharide units and are frequently polysulfated.…”

Electron detachment dissociation of dermatan sulfate oligosaccharides

“…We have recently reported the utility of electron detachment dissociation (EDD) [23][24][25][26][27] for the analysis of GAG HS tetrasaccharides [28,29]. EDD produces a radical anion, which undergoes more extensive fragmentation than that produced from activation of even electron ions by low-energy or threshold dissociation methods.…”