Franklin E. Leach

Modular Synthesis of Heparan Sulfate Oligosaccharides for Structure−Activity Relationship Studies

Although hundreds of heparan sulfate binding proteins have been identified and implicated in a myriad of physiological and pathological processes, very little information is known about the ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS oligosaccharides. To address this deficiency, we developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of 12 oligosaccharides, which has been employed to probe the structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimers disease.

The proteoglycan bikunin has a defined sequence

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan having a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike the template driven synthesis of nucleic acids and proteins, Golgi synthesized glycosaminoglycans are not believed to have predictable or deterministic sequence. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size similar and charge similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular-ions corresponding to mono-compositional peptidoglycosaminoglycans. Fragmentation using collision induced dissociation surprisingly afforded a single sequence for each mono-compositional parent-ion, unequivocally demonstrating the presence of a defined sequence. The common biosynthetic pathway for all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment of our understanding of information storage in complex glycans.

Negative electron transfer dissociation of glycosaminoglycans.

Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation. Using fluoranthene or xenon, both glycosidic and cross-ring cleavages are observed, as well as even- and odd-electron products. The loss of SO(3) can be minimized and an increase in cross-ring cleavages is observed if a negatively charged carboxylate is present during NETD, which can be controlled by the charge state or the addition of sodium. NETD effectively dissociates GAGs up to eight saccharides in length, but the low resolution of the ion trap makes assigning product ions difficult. Similar to EDD, NETD is also able to distinguish the epimers iduronic acid from glucuronic acid in heparan sulfate tetrasaccharides and suggests that a radical intermediate plays an important role in distinguishing these epimers. These results demonstrate that NETD is effective at characterizing GAG oligosaccharides in a single tandem mass spectrometry experiment on a widely available mass spectrometry platform.

Overexpression of ST6GalNAcV, a ganglioside-specific α2,6-sialyltransferase, inhibits glioma growth in vivo

Aberrant cell-surface glycosylation patterns are present on virtually all tumors and have been linked to tumor progression, metastasis, and invasivity. We have shown that expressing a normally quiescent, glycoprotein-specific α2,6-sialyltransferase (ST6Gal1) gene in gliomas inhibited invasivity in vitro and tumor formation in vivo. To identify other glycogene targets with therapeutic potential, we created a focused 45-mer oligonucleotide microarray platform representing all of the cloned human glycotranscriptome and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Among the many significant changes in glycogene expression observed, of particular interest was the observation that an additional α2,6-sialyltransferase, ST6 (α-N-acetyl-neuraminyl-2,3-β-galactosyl-1,3)-N-acetylgalactosaminide α2,6-sialyltransferase 5 (ST6GalNAcV), was expressed at very low levels in all glioma and glioma cell lines examined compared with normal brain. ST6GalNAcV catalyzes the formation of the terminal α2,6-sialic acid linkages on gangliosides. Stable transfection of ST6GalNAcV into U373MG glioma cells produced (i) no change in α2,6-linked sialic acid-containing glycoproteins, (ii) increased expression of GM2α and GM3 gangliosides and decreased expression of GM1b, Gb3, and Gb4, (iii) marked inhibition of in vitro invasivity, (iv) modified cellular adhesion to fibronectin and laminin, (v) increased adhesion-mediated protein tyrosine phosphorylation of HSPA8, and (vi) inhibition of tumor growth in vivo. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters invasivity in a manner that may have significant therapeutic potential.

Hexuronic Acid Stereochemistry Determination in Chondroitin Sulfate Glycosaminoglycan Oligosaccharides by Electron Detachment Dissociation

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated 0,2X3 and Y3 ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.

Comparison of particle-in-cell simulations with experimentally observed frequency shifts between ions of the same mass-to-charge in fourier transform ion cyclotron resonance mass spectrometry

It has been previously observed that the measured frequency of ions in a Fourier transform mass spectrometry experiment depend upon the number of trapped ions, even for populations consisting exclusively of a single mass-to-charge. Since ions of the same mass-to-charge are thought not to exert a space-charge effect among themselves, the experimental observation of such frequency shifts raises questions about their origin. To determine the source of such experimentally observed frequency shifts, multiparticle ion trajectory simulations have been conducted on monoisotopic populations of Cs+ ranging from 102 ions to 106 ions. A close match to experimental behavior is observed. By probing the effect of ion number and orbital radius on the shift in the cyclotron frequency, it is shown that for a monoisotopic population of ions, the frequency shift is caused by the interaction of ions with their image-charge. The addition of ions of a second mass-to-charge to the simulation allows the comparison of the magnitude of the frequency shift resulting from space-charge (ion-ion) effects versus ion interactions with their image charge.

Negative electron transfer dissociation Fourier transform mass spectrometry of glycosaminoglycan carbohydrates.

Electron transfer through gas-phase ion–ion reactions has led to the widespread application of electron-based techniques once only capable in ion trapping mass spectrometers. Although any mass analyzer can, in theory, be coupled to an ion–ion reaction device (typically a 3-D ion trap), some systems of interest exceed the capabilities of most mass spectrometers. This case is particularly true in the structural characterization of glycosaminoglycan (GAG) oligosaccharides. To adequately characterize highly sulfated GAGs or oligosaccharides above the tetrasaccharide level, a high-resolution mass analyzer is required. To extend previous efforts on an ion trap mass spectrometer, negative electron transfer dissociation coupled with a Fourier transform ion cyclotron resonance mass spectrometer has been applied to increasingly sulfated heparan sulfate and heparin tetrasaccharides as well as a dermatan sulfate octasaccharide. Results similar to those obtained by electron detachment dissociation are observed.

Multivariate Analysis of Electron Detachment Dissociation and Infrared Multiphoton Dissociation Mass Spectra of Heparan Sulfate Tetrasaccharides Differing Only in Hexuronic acid Stereochemistry

The structural characterization of glycosaminoglycan (GAG) carbohydrates by mass spectrometry has been a long-standing analytical challenge due to the inherent heterogeneity of these biomolecules, specifically polydispersity, variability in sulfation, and hexuronic acid stereochemistry. Recent advances in tandem mass spectrometry methods employing threshold and electron-based ion activation have resulted in the ability to determine the location of the labile sulfate modification as well as assign the stereochemistry of hexuronic acid residues. To facilitate the analysis of complex electron detachment dissociation (EDD) spectra, principal component analysis (PCA) is employed to differentiate the hexuronic acid stereochemistry of four synthetic GAG epimers whose EDD spectra are nearly identical upon visual inspection. For comparison, PCA is also applied to infrared multiphoton dissociation spectra (IRMPD) of the examined epimers. To assess the applicability of multivariate methods in GAG mixture analysis, PCA is utilized to identify the relative content of two epimers in a binary mixture.

ELECTRON CAPTURE DISSOCIATION, ELECTRON DETACHMENT DISSOCIATION, AND INFRARED MULTIPHOTON DISSOCIATION OF SUCROSE OCTASULFATE

The structural analysis of sulfated carbohydrates such as glycosaminoglycans (GAGs) has been a long-standing challenge for the field of mass spectrometry. The dissociation of sulfated carbohydrates by collisionally-activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD), which activate ions via vibrational excitation, typically result in few cleavages and abundant SO3 loss for highly sulfated GAGs such as heparin and heparan sulfate, hampering efforts to determine sites of modification. The recent application of electron activation techniques, specifically electron capture dissociation (ECD) and electron detachment dissociation (EDD), provides a marked improvement for the mass spectrometry characterization of GAGs. In this work, we compare ECD, EDD and IRMPD for the dissociation of the highly sulfated carbohydrate sucrose octasulfate (SOS). Both positive and negative multiply-charged ions are investigated. ECD, EDD and IRMPD of SOS produce abundant and reproducible fragmentation. The product ions produced by ECD are quite different than those produced by IRMPD of SOS positive ions, suggesting different dissociation mechanisms as a result of electronic versus vibrational excitation. The product ions produced by EDD and IRMPD of SOS negative ions also differ from each other. Evidence for SO3 rearrangement exists in the negative ion IRMPD data, complicating the assignment of product ions.

Glycosaminoglycan Characterization by Electrospray Ionization Mass Spectrometry Including Fourier Transform Mass Spectrometry

Electrospray ionization mass spectrometry (ESI MS) is a versatile analytical technique in glycomics of glycosaminoglycans (GAGs). Combined with enzymology, ESI MS is used for assessing changes in disaccharide composition of GAGs biosynthesized under different environmental or physiological conditions. ESI coupled with high-resolution mass analyzers such as a Fourier transform mass spectrometer (FTMS) permits accurate mass measurement of large oligosaccharides and intact GAGs as well as structural characterization of GAG oligosaccharides using information-rich fragmentation methods such as electron detachment dissociation. The first part of this chapter describes methods for disaccharide compositional profiling using ESI MS and the second part is dedicated to FTMS and tandem MS methods of GAG compositional and structural analysis.