Comparative Studies of Glycoprotein Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry
|Keywords:||醣蛋白;蛋白質濃度;醣化程度;醣鏈分佈;質譜;glycoprotein;protein concentration;glycosylation site occupancy;glycan profile;mass spectrometry||Issue Date:||2010||Abstract:||
Glycoproteins are vared with regulatory states, type of disease, and even with disease progression. Therefore, comparative studies of the change of glycoproteins may provide useful information for the diagnosis of disease. Several causes including the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile may account for the difference if the signal of a glycopeptide was found to be changed. A new strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d0 and formaldehyde-d2 along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The feasibility of the new strategy under different situations was studied as following: (1)glycoprotein concentration changed, glycan profile and glycosylation site occupancy fixed; (2)glycan profile changed, glycoprotein concentration and glycosylation site occupancy fixed; (3)glycosylation site occupancy changed, glycoprotein concentration and glycan profile fixed; (4) glycoprotein concentration, glycan profile and glycosylation site occupancy all changed. As a consequence, the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be obtained simultaneously. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS2 and MS3 of the instrument, the site of glycosylation and the glycan sequence may also be obtained in positive-ion mode. The negative-ion mode of MSn was useful for glycan structural analysis.
To test the feasibility of the proposed strategy in complex glcoprotein with more than one glycosylation site and sialylated N-glycans, lactoferrin was chosen as the model compound. Cellulose microcrystalline enrichment was replaced by reversed-phase liquid chromatography. The results showed that the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be obtained in complex glycoprotein sample.
To apply the strategy on lactoferrin from human milk of different stages of lactation period, first optimize purification method by lactoferrin standard and market milk. Lactoferrin was successfully purified form human milk. The glycoprotein change of lactoferrin from different lactation stages due to glycoprotein concentration, glycosylation site occupancy and glycan profile could be differentiated by the strategy.
|Appears in Collections:||化學系|
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