Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides

Abstract

Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points. One format of IEF, the capillary isoelectric focusing (cIEF), is an attractive approach in that it can be coupled with mass spectrometry (cIEF-MS) to provide online focusing and detection of complex mixtures. In the past three decades, numerous studies have applied cIEF-MS methods to the analysis of protein and peptide mixtures by positive-ion mode mass spectrometry. However, polysaccharide chemists largely rely on negative-ion mode mass spectrometry for the analysis of highly sulfated GAGs. The current study reports a negative-ion mode cIEF-MS method using an electrokinetically pumped sheath liquid nanospray capillary electrophoresis–mass spectrometry (CE-MS) coupling technology. The feasibility of this negative-ion cIEF-MS method and its potential applications are demonstrated using chondroitin sulfate and heparan sulfate oligosaccharides mixtures.

Challenging Bioanalyses with Capillary Electrophoresis

“…Structural and chemical information was obtained for the anticoagulant heparin drug, enoxaparin, which contained a mixture of low molecular weight heparins as well as isomeric structures. In a different report, capillary isoelectric focusing was used to separate mixtures of heparan sulfate and chondroitin sulfate . Glycosaminoglycans were focused under reverse polarity according to subtle differences in isoelectric point, which was related to the level of sulfation in different structures.…”

“…In a different report, capillary isoelectric focusing was used to separate mixtures of heparan sulfate and chondroitin sulfate. 154 Glycosaminoglycans were focused under reverse polarity according to subtle differences in isoelectric point, which was related to the level of sulfation in different structures.…”

Recent advances in biotechnology for heparin and heparan sulfate analysis

“…Although a CE-MS interface offers a rapid analytical method and has been commercialized, the sensitivity and reproducibility of this method needs further improvement. Online reversepolarity negative-ion mode capillary isoelectric focusing (cIEF)-MS based on an electrokinetically pumped sheath liquid nanospray CE-MS for the separation and detection of HS disaccharides (Figure 6C) [53]. Using a high voltage a pH gradient with low pH at the anode and high pH at the cathode is formed within the capillary.…”

Glycan Mapping of Low-Molecular-Weight Heparin Using Mass Spectral Correction Based on Chromatography Fitting with “Glycomapping” Software

“…A combination of different separation techniques with mass spectrometry (MS) 8,14,15 has been widely used by analytical chemists to address these questions. These hyphenated techniques include reverse phase chromatography ion-pairing (RPIP)-MS, hydrophilic interaction liquid chromatography (HILIC)-MS, size exclusion chromatography (SEC)-MS, and capillary electrophoresis-MS. 16 RPIP 17,18 has the highest chromatographic resolution for the complex molecules in LMWHs but cannot easily rely on ultraviolet (UV) detection. Moreover, ion pairing reagent often remains in the mass spectrometer resulting in analysis instability.…”