Developments in Mass Spectrometry for Glycosaminoglycan Analysis: A Review.

This review covers recent developments in glycosaminoglycan (GAG) analysis via mass spectrometry (MS). GAGs participate in a variety of biological functions, including cellular communication, wound healing, and anticoagulation, and are important targets for structural characterization. GAGs exhibit a diverse range of structural features due to the variety of O- and N-sulfation modifications and uronic acid C-5 epimerization that can occur, making their analysis a challenging target. Mass spectrometry approaches to the structure assignment of GAGs have been widely investigated, and new methodologies remain the subject of development. Advances in sample preparation, tandem MS techniques (MS/MS), online separations, and automated analysis software have advanced the field of GAG analysis. These recent developments have led to remarkable improvements in the precision and time efficiency for the structural characterization of GAGs.

Investigation of electrospray for a capillary electrophoresis-mass spectrometry interface in reverse polarity and negative ion mode.

Capillary zone electrophoresis (CZE) paired with mass spectrometry (MS) is a powerful analytical technique for examining mixtures of ionic analytes such as glycosaminoglycans. This study examines the mechanics of the electrospray process for a sheath flow CZE-MS interface under reverse polarity negative ionization conditions. Liquid flow in a sheath flow nano-electrospray CZE-MS interface is driven by two mechanisms, electroosmotic flow and electrospray nebulization. The contribution of these two processes to the overall flow of solution to the electrospray tip is influenced by the surface coatings of the sheath flow emitter tip and by the solvent composition. We have investigated the application of this interface to the reverse polarity separation of glycosaminoglycans and find that the role of electroosmotic flow is far less than has been reported previously, and the electrospray process itself is the largest contributor to the flow of the sheath liquid.

Online Capillary Zone Electrophoresis Negative Electron Transfer Dissociation Tandem Mass Spectrometry of Glycosaminoglycan Mixtures.

Glycosaminoglycans (GAGs) are important biological molecules that are highly anionic and occur in nature as complex mixtures. A platform that combines capillary zone electrophoresis (CZE) separations with mass spectrometry (MS) and gas-phase sequencing by using negative electron transfer dissociation (NETD) is shown to be efficacious for the structural analysis of GAG mixtures. CZE is a separation method well suited to the highly negatively charged nature of GAGs. NETD is an electron-based ion activation method that enables the generation of informative fragments with retention of the labile sulfate half-ester modification that determine specific GAG function. Here we combine for the first time NETD and CZE for assigning the structures of GAG oligomers present in mixtures. The speed of ion activation by NETD is found to couple well with the narrow peaks resulting from CZE migration. The platform was optimized with mixtures of GAG tetrasaccharide standards. The potential of the platform is demonstrated by the analysis of enoxaparin, a complex mixture of low molecular weight heparins, which was separated by CZE within 30 minutes and characterized by NETD MS/MS in one online experiment. 37 unique molecular compositions have been identified in enoxaparin using CZE-MS and 9 structures have been assigned with CZE-NETD-MS/MS.

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement.

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.

An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.