| Glycoprotein is involved in all of life events as a kind of key component and functional material in biolog.There are two main types of glycosylation:N-glycosylation and O-glycosylation,which corresbonding two kinds of glycoprotein oligosaccharides:N-glycan and O-glycan.Althrough universally analytical methods of those two types of glycoprotein oligosaccharides have been found,they still have disadvantages of structural analysis for no matter N-or O-glycan.This article optimized currently analycal method of N-or O-glycans structure,and used the optimized method to analysis the structure of two type of glycoprotein oligosaccharide from food.1.The optimization and application of analysis method of N-glycan structure from foodOligosaccharide should be released from glycoprotein before being analyzed,and there are two kinds of common tools to release N-glycan from glycoprotein:PNGase F and PNGase A.However,both of them have disadvantages.A novel PNGase enzyme encoded gene was discovered from the bacterial strain Terriglobus roseus DSM 18391 in our laboratory,and it was recombinant expressed in prokaryotic system.Recombinant PNGase H+ could not only digest all kinds of N-glycosylation no matter its substrate is glycopeptides or glycoprotein,but also won't pollute the products because it isn't glycoprotein itself.Based on PNGase H+,reagents and processes are optimized to build a faster and more convenient method of glycosylation.Because this enzyme is highly active under acidic conditions,the consecutive fluorescence labeling step using 2-aminobenzamide(2-AB)can be directly performed in the same mixture used for the enzymatic deglycosylation step,which greatly reduces sample handling,reaction and detection time.This methodology was verified using HRP as a model glycoprotein and quantified the major HRP N-glycan MMXF3.It is found that 1 mol/L acetic acid is the optimum conditions in which the glycoprotein can be fully digested.Combined with HPLC,all sample handling and incubation steps can be performed in less than 4 h without the need for tedious purification and vacuum rotary drying.Compared with traditional method,the optimized method will reduce the loss of samples.And the stability and applicability are verified through the different concentrations of substrate.According to this rapid method,we successfully analyzed the N-glycan derived from various plantsa and mushrooms in deep through using ultra-performance liquid chromatography(UPLC)and MALDI-ToF-MS spectrum.2.The optimization and application of analysis method of O-glycan structure from foodCompared to N-glycan,the structure of O-glycan is too complex to allow one kind of enzyme to release all types of O-glycan from glycoprotein just like Pongees by now,and chemical way is the only method to choose.One kind of chemical method called new ?-elimination arise attention recently,and this ammonia based ?-elimination has mild reaction conditions while it has good effects on releasing O-glycan.However,the peeling reaction still exists.Reactive temperature and time are comprehensively optimized in this experimental,and it is found that the total yield of O-glycans is as high as possible and the peeling reaction is as low as possible when it reacts 16 hours on 60?.Combining with HPLC,optimized ?-elimination method is used in this article to analyze the structure of O-glycan which plays significantly biological role in milk,and relatively good results of HPLC peak and structure were gotten. |
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