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摘要
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Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). After being degraded by chondroitinase ABC, chondroitin sulfate disaccharide containing an unsaturated double bond (CS-disaccharide) is obtained. In this study, we investigated the effect of a high salt culture medium on the enzymatic degradation reaction and the detection signal of CS-disaccharide by matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry (MALDI-TOF/MS). The high salt content of the culture medium caused the MALDI samples that mixed CS disaccharide and 2,5-dihydroxybenzoic acid (DHB) matrix to be covered with thick salt crystals. As a result, the CS-disaccharide could not be detected. After diluting the sample ten times, the salt interference and the CS-disaccharide content decreased. The CS-disaccharide signal could be detected, but the intensity was still too low. Taking advantage of the negative charge characteristics of the carboxylate and sulfate groups on the CS-disaccharide, the CS-disaccharide signal could be significantly improved using solid sorbent to desalt the sample. We have studied several factors that would affect the salt removal efficiency, including the type, the usage amount, and the activation method of the adsorbent. It was found that adsorbents such as the acid-activated Yttria-stabilized zirconia (YSZ) and the alkali-activated chitosan had desalting capabilities and could enhance the intensity of CS-disaccharide signals. Desalted by high-DDA chitosan, very high CS-disaccharide signals could be detected in both RP and RN modes of MALDI-TOF/MS. By adjusting the usage amount and the activation method of chitosan, optimal desalting conditions could be achieved, further improving the sensitivity of MALDI-TOF/MS in detecting CS-disaccharide in a high salt culture medium. |