Delineating mechanisms of dissociation for isomeric heparin disaccharides using isotope labeling and ion trap tandem mass spectrometry

Abstract

Heparin and heparan sulfate (HS) glycosaminoglycans have been identified as important players in many physiological as well as pathophysiological settings. A better understanding of the biosynthesis and structure of these molecules is critical for further elucidation of their biological function. We have demonstrated the successful use of negative electrospray ionization tandem mass spectrometry in the differentiation of all twelve standard heparinbuilding blocks, including the potentially important N-unsubstituted disaccharides. Collision induced dissociation of each of the isomeric disaccharides provided unique product ion spectra, useful for identification and quantification of the relative amounts of each isomer present. In the research presented herein, isotopic labeling studies using 18 O and 2 H were used to determine the origins of each of the neutral losses observed in the product ion spectra, and mechanisms of dissociation consistent with the observed data were postulated. The general mechanisms postulated were for the generation of B, Y, and Z ions formed from glycosidic cleavages, as well as A and X ions formed from cross-ring cleavages. The eight isomeric heparin disaccharides all underwent cross-ring cleavage to form 0,2 X 1 and 0,2 A 2 ions, and further experiments suggest that the mechanisms of formation of these ions are through a charge-remote process. The tandem mass spectrometry data presented herein also provide a foundation for further developments towards a practical analysis tool for the structural elucidation of larger, biologically important heparin/HS oligosaccharides by using mass spectrometry. H eparin and heparan sulfate (HS) glycosaminoglycans (GAGs) have been implicated in a host of biological functions including blood coagulation, inflammatory processes, cell-cell and cellmatrix interactions, as well as cell growth and differentiation [1]. Although heparin has been used clinically as an anticoagulant for decades, and the closely related species, heparan sulfate has been identified as a key player with many biological roles, the absolute structures responsible for the various attributed functions are complex and have only recently been deciphered in a few specific cases. To better understand their biological roles in physiological as well as pathophysiological settings, it is necessary to gain a better understanding about the structure of heparin/HS at the molecular level.Heparin/HS polysaccharides consist of a repeating disaccharide unit of [HexA␤(1,4)GlcN␣(1,4)] n . The hexuronic acid (HexA) residue can be either L-iduronic acid (IdoA) or D-glucuronic acid (GlcA) depending on the stereochemistry of the C5 carboxylic acid group, and may be sulfated at the C2 position. In addition, the glucosamine units may be N-acetylated (GlcNAc), Nsulfated (GlcNS) or N-unsubstituted (GlcN), and can be further differentially sulfated at the C3 and C6 positions. Structural studies of heparin and HS have indicated differences between the two, such as the predominance of the trisulfated disaccharid...

Wavelength-tunable ultraviolet photodissociation (UVPD) of heparin-derived disaccharides in a linear ion trap

“…They are similar to those previously reported by Saad and Leary [25]. Figures 3b, 4b, and 5b provide their corresponding CID spectra.…”

ESI-MS differential fragmentation of positional isomers of sulfated oligosaccharides derived from carrageenans and agarans

“…Ϫ ions with higher relative intensities than did G4S-AOH (3) and G3S-AOH (8), which can also explain the lower relative abundance of [M-Na] Ϫ for those oligosaccharides that had greater intensities for loss of sulfate (see Table 2). Despite this fact, the glycosidic cleavage observations are consistent with the previously proposed [20] sulfate-mediated hydrogen transfer for B 1 formation; we then concluded that this mechanism justifies the observation that the two oligosaccharides having the sulfate group spatially closer to the glycosidic linkage, G2S-AOH (6) and G6S-AOH (7), undergo an easier B 1 fragmentation. This supposition is also supported by glycosidic fragmentation patterns found for G4S-A-G4S-AOH (4) and G4S-A-G4S-A-G4S-AOH (5).…”

“…To elucidate the origin of these fragment ions, we first attempted to establish the probable structure of the precursor B 1 ion. Along with glycosidic bond cleavage, previously general B-type cleavage mechanisms propose: (a) epoxide formation [56]; (b) double bond formation (between C-1 and C-2) with ring opening [20], and (c) double bond formation without ring opening [56]. The B 1 formation mechanism that better explains most of our results corresponds to (c) as shown in Scheme 1 [56].…”

Electron detachment dissociation of glycosaminoglycan tetrasaccharides

“…The singly-charged even-electron products that arise from direct fragmentation of the doublycharged negative ion can be identified by using IRMPD or CAD for ion activation, as their ions are produced by dissociation of an even-electron precursor. IRMPD of 1 produces°the°mass°spectrum°shown°in°Figure°2b,°while CAD°of°1 produces°the°mass°spectrum°shown°in°Figure 1c.°The°major°fragments°in°Figure°2b°and°c°are°princi-pally from glycosidic bond cleavages (B, C, Y, and Z) and a few cross ring cleavages in the form of 0,2 A 4 and 40].°All°of°the°fragments°observed°in°the°CAD°spectrum are found in the IRMPD spectrum, and all the IRMPD products°are°present°in°the°EDD°spectrum°(Figure°2, insets). We have found this to be generally true for all of the GAG tetrasaccharides examined to date, i.e., EDD gives the most comprehensive set of fragment ions, while the IRMPD products are a subset of the ions in the EDD spectrum, and the CAD products are a subset of the ions in the IRMPD spectrum.…”