Differentiating Chondroitin Sulfate Glycosaminoglycans Using Collision-Induced Dissociation; Uronic Acid Cross-Ring Diagnostic Fragments in a Single Stage of Tandem Mass Spectrometry

Abstract

The stereochemistry of the hexuronic acid residues of the structure of glycosaminoglycans (GAGs) is a key feature that affects their interactions with proteins and other biological functions. Electron based tandem mass spectrometry methods, in particular electron detachment dissociation (EDD), have been able to distinguish glucuronic (GlcA) from iduronic acid (IdoA) residues in some heparan sulfate tetrasaccharides by producing epimer-specific fragments. Similarly, the relative abundance of glycosidic fragment ions produced by collision induced dissociation (CID) or EDD have been shown to correlate with the type of hexuronic acid present in chondroitin sulfate (CS) GAGs. The present work examines the effect of charge state and the degree of sodium cationization on the CID fragmentation products that can be used to distinguish GlcA and IdoA containing chondroitin sulfate A (CSA) and dermatan sulfate (DS) chains. The cross-ring fragments 2,4An and 0,2Xn formed within the hexuronic acid residues are highly preferential for chains containing GlcA, distinguishing it from IdoA. The diagnostic capability of the fragments requires the selection of a molecular ion and fragment ions with specific ionization characteristics, namely charge state and the number of ionizable protons. The ions with the proper characteristics display diagnostic properties for all the CS and DS chains (dp4-dp10) studied.

Isomer separation and effect of the degree of polymerization on the gas‐phase structure of chondroitin sulfate oligosaccharides analyzed by ion mobility and tandem mass spectrometry

“…Different studies have reported the comprehensive MS characterization of the various sulfated dp2 isomers from glucosaminoglycans allowing decisive progresses in the compositional analysis and profiling of heparin and heparan sulfate from multiple origins . Less data are comparatively available for galactosaminoglycan‐derived oligosaccharides, especially as regards the detailed characterization of chondroitin sulfate oligosaccharides . Previous studies showed that MS/MS fragmentation of CS‐A and CS‐C dp2 produced identical Y 1 and Z 1 fragments with different respective intensities .…”

“…[19][20][21][22][23][24] Less data are comparatively available for galactosaminoglycan-derived oligosaccharides, especially as regards the detailed characterization of chondroitin sulfate oligosaccharides. [25][26][27][28][29][30] Previous studies showed that MS/MS fragmentation of CS-A and CS-C dp2 produced identical Y 1 and Z 1 fragments with different respective intensities. 31,32 Therefore, the determination of the 4S/6S sequence of CS could not rely unambiguously on these fragment ions only, but must also involve the measurement of their intensity or further multistage (MS n ) collision-induced dissociation.…”

Applications of capillary electrophoresis electrospray ionization mass spectrometry in glycosaminoglycan analysis

“…In the last decade, ESI MS contributed a significant progress to compositional and structural analysis of glycosaminoglycans (GAGs) with a particular emphasis on the determination by advanced MS/MS techniques of chain sequences, the complex sulfation patterns, and their correlation with certain biological activities . However, ESI MS has encountered a number of impediments, the most severe being the difficulty to ionize long GAG chains, prevent the in‐source fragmentation of the labile sulfate groups, generate multiply charged ions of high molecular weight (MW) species, simultaneously ionize and detect chains of variable sulfation degree, and distinguish the isobaric structures.…”

Single Stage Tandem Mass Spectrometry Assignment of the C-5 Uronic Acid Stereochemistry in Heparan Sulfate Tetrasaccharides using Electron Detachment Dissociation

“…Previous EDD and CID reports have shown the presence of sodium counter ions can provide a means to control stereochemical dependent fragmentation [21, 36]. We found that to be very useful for the tri and tetra-sulfated HS standards.…”

“…[35]. The ability to assign the C-5 stereochemistry in uronic acid residues of 4- O -sulfo chondroitin sulfate epimers with varying oligomer lengths (dp4–dp10) based on diagnostic cross-ring ions 2,4 A n , and 0,2 X n in CID mass spectra has also been reported by Kailemia et al [36]. Compared to heparan sulfates, chondroitin sulfates have well-defined sulfation pattern, hence the former requires a more sensitive activation method for stereochemistry assignments.…”