Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by chip-based nanoelectrospray multistage mass spectrometry

Abstract

Sulfation pattern within chondroitin sulfate (CS) glycosaminoglycan (GAG) chains is an important post-translational modification that regulates their interaction with proteins. In this context, development of highly efficient and reproducible analytical methods for the investigation of CS sulfation patterns is of high necessity. In this study we report a novel method for straightforward determination of N-acetylgalactosamine (GalNAc) sulfation sites in chondroitin sulfate disaccharides. Our protocol involves combining fully automated chip-based nanoelectrospray (nanoESI) for analyte infusion and ionization in negative ion mode with multistage (MS n ) collision-induced dissociation (CID) high capacity ion trap (HCT) mass spectrometry for generation of sequence ions diagnostic for identification of sulfate ester group position within GalNAc residues. The feasibility of this approach is here demonstrated on chondroitin 6-O-sulfate and chondroitin 4-O-sulfate disaccharides. Fragmentation patterns obtained by MS 2 and MS 3 sequencing stages provided first mass spectrometric data from which sulfation site(s) within GalNAc monosaccharide ring could be unequivocally deciphered. Hence, the method allowed discriminating 4S/6S sulfation sites solely on the basis of MS and multistage MS evidence. Warsaw and Springer-Verlag Berlin Heidelberg. © Versita

Chip‐based high resolution tandem mass spectrometric determination of fibroblast growth factor—chondroitin sulfate disaccharides noncovalent interaction

“…Given the ergodic nature of CID process, rearrangements in the collision cell, implying SO 3 − migration, were observed in some instances and were reported by McClellan et al and Kenny et al However, our previous MS/MS experiments performed on commercially available CS/DS having the sulfate position specified by the producer have shown that, by applying the fragmentation protocol developed by us , , , which was also used in the present study, such rearrangements and SO 3 − migration did not occur in CID MS/MS.…”

Combining size‐exclusion chromatography and fully automated chip‐based nanoelectrospray quadrupole time‐of‐flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

“…Remarkably, though the feasibility in GAG analysis of chip-based nanoESI could be demonstrated before only on CS/DS disaccharides [25,26], by the present protocol, spectra of elevated signal-to-noise ratio could be produced even for longer CS/DS chains, within only a few minutes of signal acquisition and with considerable reduction in sample consumption. Thus, in these experiments, the analysis sensitivity was situated in the low picomole range.…”

“…The latter process was controlled by decreasing the voltages on the pipette tip and cone until transfer into MS of multiply charged anions, corresponding to deprotonation of all sulfate groups was observed. This set of optimal parameters was assessed by us previously on standard 4-sulfate and 6-sulfate CS disaccharides [25]. Following sample infusion and MS analysis, the pipette tip was ejected Electrophoresis 2011Electrophoresis , 32, 1639Electrophoresis -1646 and a new tip and nozzle were used for each sample, thus preventing any cross-contamination or carry-over.…”

“…By this method, regularly sulfated CS/DS chains, up to hexamers, were identified and thoroughly characterized but no oversulfated sequences were discovered. Due to the technical advancements in MS instrumentation, in particular of the systems able to rapidly perform multistage MS (MS n ) by either collision-induced dissociation (CID) [11,18,21,25,26,37] or electrondetachment dissociation (EDD) [38,39] and the development of consistent sequencing protocols, nowadays, the determination of CS/DS structure in terms of chain length, epimerization, sulfation global content, sulfation pattern and site(s) is also feasible.…”

IRMPD Spectroscopy Sheds New (Infrared) Light on the Sulfate Pattern of Carbohydrates

“…These two disaccharides derived from CS possess notoriously ambiguous MS/MS signatures with variable ratios of Z and Y ions depending on the experimental conditions, [33][34][35] it is hence challenging to identify them by MS/MS. Additionally, the IRMPD spectra of unsaturated disaccharides derived from Hp are distinct from their isomers derived from CS.…”