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

Abstract

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)⁴] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]⁴ ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)₃](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.

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

“…The ion assignment to species containing 4,5‐Δ‐GlcA was based on the mechanism of chondroitin AC lyase cleavage reaction, forming a 4,5 double bond in the GlcA residue. According to previous observations, chondroitin AC lyase cleaves the 3GalNAcβ1‐4GlcAβ1 glycosidic bond, irrespective of the sulfation content, distribution along the chain and/or the site(s) in the oligosaccharide ring.…”

“…After the introduction of microfluidics for ESI, the capability of MS in the glycosaminoglycomics, in particular, in CS/DS research, increased remarkably. For this reason, the NanoMate robot encompassing automatic sample infusion using nanoESI was coupled to different types of instruments for the identification and structural characterization of CS/DS, such as the high‐capacity ion trap (HCT) or the quadrupole time‐of‐flight (QTOF) . This method, together with a fragmentation technique such as collision‐induced dissociation tandem MS (CID MS/MS) proved its feasibility in discovering and structural characterization of novel molecular species with a potential biomarker role in various biological matrices as shown by our previous work .…”

Identification of an unusually sulfated tetrasaccharide chondroitin/dermatan motif in mouse brain by combining chip‐nanoelectrospray multistage MS2‐MS4 and high resolution MS

“…This cleavage is independent of sulfation pattern and content of GalNAc and GlcA ; therefore all GalNAc–GlcA sequences represent substrates prone to be cleaved by the enzyme, even under nonexhaustive digestion conditions. In view of this aspect, demonstrated previously , it appears obvious that the chains longer than disaccharide contain GalNAc–IdoA linkages for which Chondroitinase AC I Flavo has no specificity. Even if the stereochemistry of the hexuronic acid from the nonreducing end was lost, for a better insight into the structure of these complex molecules we kept the original annotation due to the known origin of GlcA.…”

“…Recently, a chip‐based nanoelectrospray system (NanoMate robot) in direct coupling with either high‐capacity ion trap (HCT) or QTOF mass analyzers was successfully applied to CS/DS screening and sequencing at elevated sensitivity, reproducibility, and throughput. In our previous study on chip‐nanoESI MS n feasibility in brain GAG research, a new oversulfated 4,5‐Δ‐[GlcA(3S)‐GalNAc(6S)] motif was for the first time identified in a neural tissue.…”

Decorin in atherosclerosis

“…Each molecule of decorin can have either one or two chains of either of these glycosaminoglycans [Zamfir et al 2011].…”