| First of all, the paper was establishing methods of HPLC-PDA and HPLC-ESI-MS(MS/MS) to analyze the products of Chondroitin sulfate (CS of pig) derived from completely and partially enzymatic degradation. The optimization of conditions of sample preparation and HPLC-MS/MS was conducted. Three MS methods for analyzing and identifying the products derived from enzymatic degradation of CS were conducted and compared. Then, the developed LC-ESI-MS/MS method was applied to detect and analyze the products derived from enzymatic degradation of Salmon and Snakehead. Finally, HA and CS were chemically degraded by Fenton reagents and the oligosaccharides derived from chemical degradation was analyzed by HPLC-MS and compared with the oligosaccharides derived from enzymatic degradation.Firstly, methods for analyzing and identifying products derived from completely enzymatic degradation of CS (pig) by HPLC-PDA and HPLC-ESI-MS were established. The optimization of conditions of sample preparation was conducted and the optimal conditions of completely enzymatic degradation were that:the concentration of substrate was2mg/mL; concentration of enzyme was320m IU/mL; reaction time was24h; reaction buffer was phosphate buffered solution with pH=7.0. Results of LC-MS analysis showed that four kinds of disaccharide with different number and substituent sites of SO3were identified. Propotion of each disaccharide (accounting for the proportion of total disaccharide) were△Di-OS (30.1%),△Di-6S (18.5%),△Di-4S (44.9%),△Di-diSB (6.5%). The results implied that the main components of CS (pig) were CS-A and CS-B, and the content of CS-A was higher than CS-C.Then, methods for analyzing and identifying products derived from partially enzymatic degradation of CS (pig) by HPLC-ESI-MS(MS/MS) were established. The optimization of conditions of selecting chromatographic column, composition of mobile phase, and parameters of ion source and mass analyzer was conducted. Results showed that using XAmide column as chromatographic column, ACN and10mM NH4Ac as mobile phase, products of enzymatic degradation of CS could obtain6even-numbered CS oligosaccharides, whose degree of polymerization (dp) ranging from dp2to dp12. After ESI-MS/MS analysis and structural characterization of mixtures of CS-derived oligosaccharides, structures of these fragments were identified as follows: dp2:GlcAS1-GalNAcS1; dp4:dGlcAS1-GalNAcS1-GlcAS1-GalNAcS1; dp6:dGlcAS-GalNAcS1-GlcAS1-GalNAcS1-GlcA-GalNAcS1; dp8: dGlcA-S1-GalNAcS1-GlcAS1-GalNAcS1-GlcA-GalNAcS1-GlcA-GalN AcS1; dp10:dGlcA-GalNAc-(GlcA-GalNAc)4.Subsequently, three different MS methods (ESI-4000QTrap-MS/MS, ESI-Orbitrap-FT-MS/MS, MALDI-TOF-MS) for analyzing and identifying the products derived from enzymatic degradation of CS were investigated. Results implied that FT-MS/MS could identify CS oligosaccharides ranging from dp2to dp24without multiple adducts of sodium. Fragments of CS oligosaccharides in MS and MS2are very clear and abundant without any interference of impurity, and this make the analyzing and identifying structural information of oligosaccharides become easier. However, money-consuming and needing extensive maintenance makes the application of FT-MS limited. Analyzing GAGs by MALDI-TOF-MS can be handled very easily, a high sample throughout is possible, there are nearly exclusively singly charged ions and MALDI tolerates impurities and salts to a higher extent than ESI-MS. Nevertheless, MS information of analytes can be interfered with matrices used in MALDI. In addition, not using on-line analysis makes the application of MALDI limited. In our experiments, MALDI-TOF-MS can identify CS oligosaccharides up to dpl2. Small fragments of oligosaccharides such as dp4and dp6can be identified clearly, and adducts of sodium are very abundant. Sometimes it can add4to5sodium. Big fragments such as dp10and dp12can’t be identified clearly because of its weak intensity and impurity interference. Analyzing GAGs by4000QTrap MS/MS can obtain a lot of structural information, including some adducts of [M-nH]n" and [M+mNa-nH](n"m)’, but when analyzing MS2of oligosaccharides, the fragmentation information of MS2is much less compared to FT-MS. Although resolution (R) and the mass analyzer of4000QTrap MS is not as good as FT-MS, the price and availability of4000QTrap is much better than FT-MS.4000QTrap MS can satisfy the general requirements of oligosaccharides analysis (dp ranging from2to nearly16).Afterwards, the oligosaccharides derived from enzymatic degradation of CS (extracted from salmon and snakehead by our lab) were analyzed and identified by the established LC-ESI-MS/MS method. The structures of these CS oligosaccharides were compared with CS extracted from pig, whose results showed that the fragmentation sequence information of oligosaccharides with the same dp derived from three kinds of CS was very similar, except for very few different fragments. The results also implied that the number of SO3in oligosaccharides with the same dp in salmon was more than that of snakehead and pig, and the order of relative amount was:salmon>pig> snakehead.Finally, the chemical degradation products of HA and CS (pig) were investigated by HPLC-ESI-MS (4000QTRAP-MS). The HA oligosaccharides obtained by free radical depolymerization have a dp ranging from2to21, including even-and odd-numbered oligosaccharides. The LC-MS data showed that CS oligosaccharides obtained by free radical depolymerization have a dp ranging from2to12, including even-and odd-numbered oligosaccharides. Compared with enzymatic products, products of radical depolymerization are more complex that containing both even-and odd-numbered, both saturated and unsaturated oligosaccharides, but products of enzymatic degradation only found even-numbered oligosaccharides. TIC of enzymatic products is cleaner and the separation of pesks is better than radical depolymerization. Enzymatic products are regular and site specifically cleavage, however radical depolymerization is randomly fragmentated, which leads to complex products. |
PDF Full Text Request