Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides

Abstract

Liquid chromatography/mass spectrometry (LC/MS)is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversedphase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of ؊32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10,000 Da and a net charge of ؊60.The structural elucidation of complex carbohydrates remains one of the most difficult challenges for chemists, often requiring the application of multiple analytical approaches (1-5). Glycosaminoglycans (GAGs), 1 and heparin in particular, have proven to be extremely difficult to analyze because of high negative charge, polydispersity, and sequence heterogeneity (6, 7). Heparin and low molecular weight heparins, prepared through the controlled chemical or enzymatic fragmentation of heparin (8), are widely used as clinical anticoagulants. Despite their medical importance, these drugs are relatively uncharacterized in terms of their chemical structure. Moreover, heparin and the structurally related heparan sulfate exhibit many additional biological activities, making them of great interest in new drug discovery (9). Numerous challenges can arise from the structural investigation of biologically active heparin oligosaccharides, particularly those in recognition systems involving specific protein-carbohydrate interactions (10, 11). Such biologically important oligosaccharides often contain rare sequences (12, 13) and are present only in minute, often picomole quantities. New derivatization methods (14, 15), chromatography (15, 16), electrophoresis-ba...

Characterizing the Non-reducing End Structure of Heparan Sulfate

“…Great technical progress has been made in HS sequencing and structural determination in recent years (21,24,26,(31)(32)(33), but HS whole chain sequencing has not been attempted yet. The identification of the NRE of HS may open a door to this avenue.…”

Thin chip microsprayer system coupled to quadrupole time-of-flight mass spectrometer for glycoconjugate analysis

“…23,24 In particular, the assignment of already known or previously unknown components in biological mixtures is easier due to a lower degree of heterogeneity caused by cation attachment to molecular and/or fragment ions. [23][24][25][26] After acquisition, the spectra were calibrated using sodium iodide as a calibrant.…”

Determinação de heparina fracionada em preparações farmacêuticas utilizando turbidimetria

“…Entretanto, os estudos são dirigidos para a determinação das várias sequências sacarídicas que compõem a molécula e utilizam técnicas como ressonância magnética nuclear, espectrometria de massas e sistemas eletroforéticos. [8][9][10][11][12][13] Estes métodos são trabalhosos e se tornam inviáveis para a quantificação da heparina em formas farmacêuticas, pois envolvem processos de derivatização, incluindo a utilização de enzimas e a cromatografia de exclusão para a separação das diferentes frações sacarídicas. A United States Pharmacopeial Convention publicou em 2008 uma monografia atualizada para a HF, porém não descreve metodologia menos laboriosa.…”