Analysis of novel over- and under-sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS.
We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MS(n)). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MS(n). MS(2) on bisulfated 4,5-Delta-HexAGalNAc revealed an additional sulfate ester group at 4,5-Delta-HexA. MS(2) data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-Delta-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS(1) mode indicated direct correlation between the sulfate distribution and HexA epimerization. MS(n) performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)], trisulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)] with IdoA-derived 4,5-Delta-HexA at the nonreducing end, tetrasulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] and monosulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] with GlcA-derived 4,5-Delta-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.
Zamfir
Alina D
AD
Department of Chemistry and Biology, Aurel Vlaicu University of Arad, Arad, Romania.
Flangea
Corina
C
Sisu
Eugen
E
Serb
Alina F
AF
Dinca
Nicolae
N
Bruckner
Peter
P
Seidler
Daniela G
DG
D002240:Carbohydrate Sequence; D002808:Chondroitin Lyases / Q000378:metabolism*; D058575:Decorin; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification; D006025:Glycosaminoglycans / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011509:Proteoglycans / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013455:Sulfur / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011509:Proteoglycans; D013455:Sulfur; D002808:Chondroitin Lyases