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Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析 被引量:9
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作者 徐莎 张萍 +1 位作者 黄琳娟 王仲孚 《高等学校化学学报》 SCIE EI CAS CSCD 北大核心 2010年第10期1992-1998,共7页
建立了一种用非特异性酶链酶蛋白酶E(Pronase E)从糖蛋白上释放N-糖链的方法.以牛胰核糖核酸酶B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料,用Pronase E代替N-糖苷酶F(PNGase F)释放N-糖链.当蛋白酶质量与糖蛋白质量比为1∶1时,得到只... 建立了一种用非特异性酶链酶蛋白酶E(Pronase E)从糖蛋白上释放N-糖链的方法.以牛胰核糖核酸酶B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料,用Pronase E代替N-糖苷酶F(PNGase F)释放N-糖链.当蛋白酶质量与糖蛋白质量比为1∶1时,得到只带一个天冬氨酸(Asn)的闭环N-糖链,称其为糖氨酸(gly-can-Asn),这样既为糖链引入了天然的-NH2活性基团,同时还保持了糖链原有的还原端闭环结构.以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生,采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析,建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法,该方法保持了N-糖链的天然结构,便于以-NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究. 展开更多
关键词 pronase E 糖蛋白 9-氯甲酸芴甲酯 电喷雾质谱 荧光标记
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Effect of pronase as mucolytic agent on imaging quality of magnifying endoscopy 被引量:22
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作者 Gwang Ha Kim Yu Kyung Cho +2 位作者 Jae Myung Cha Sun-Young Lee Il-Kwun Chung 《World Journal of Gastroenterology》 SCIE CAS 2015年第8期2483-2489,共7页
AIM:To investigate the efficacy of premedication with pronase,a proteolytic enzyme,in improving imagequality during magnifying endoscopy.METHODS:The study was of a blinded,randomized,prospective design.Patients were a... AIM:To investigate the efficacy of premedication with pronase,a proteolytic enzyme,in improving imagequality during magnifying endoscopy.METHODS:The study was of a blinded,randomized,prospective design.Patients were assigned to groups administered oral premedication of either pronase and simethicone(Group A)or simethicone alone(Group B).First,the gastric mucosal visibility grade(1-4)was determined during conventional endoscopy,and then a magnifying endoscopic examination was conducted.The quality of images obtained by magnifying endoscopy at the stomach and the esophagus was scored from1 to 3,with a lower score indicating better visibility.The endoscopist used water flushes as needed to obtain satisfactory magnifying endoscopic views.The main study outcomes were the visibility scores during magnifying endoscopy and the number of water flushes.RESULTS:A total of 144 patients were enrolled,and data from 143 patients(M:F=90:53,mean age 57.5 years)were analyzed.The visibility score was significantly higher in the stomach following premedication with pronase(73%with a score of 1 in Group A vs 49%in Group B,P<0.05),but there was no difference in the esophagus visibility scores(67%with a score of 1in Group A vs 58%in Group B).Fewer water flushes[mean 0.7±0.9 times(range:0-3 times)in Group A vs 1.9±1.5 times(range:0-6 times)in Group B,P<0.05]in the pronase premedication group did not affect the endoscopic procedure times[mean 766 s(range:647-866 s)for Group A vs 760 s(range:678-854 s)for Group B,P=0.88].The total gastric mucosal visibility score was also lower in Group A(4.9±1.5 vs 8.3±1.8in Group B,P<0.01).CONCLUSION:The addition of pronase to simethicone premedication resulted in clearer images during magnifying endoscopy and reduced the need for water flushes. 展开更多
关键词 pronase NARROW-BAND imaging EndoscopyImage ENHANCE
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Effects of premedication with Pronase for endoscopic ultrasound of the stomach: A randomized controlled trial 被引量:8
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作者 Guo-Xin Wang Xiang Liu +3 位作者 Sheng Wang Nan Ge Jin-Tao Guo Si-Yu Sun 《World Journal of Gastroenterology》 SCIE CAS 2016年第48期10673-10679,共7页
AIM To analyze the effects of premedication with Pronase for endoscopic ultrasound(EUS) examination of the stomach.METHODS This was a prospective, randomized and controlled clinical study. All patients were randomly a... AIM To analyze the effects of premedication with Pronase for endoscopic ultrasound(EUS) examination of the stomach.METHODS This was a prospective, randomized and controlled clinical study. All patients were randomly assigned to either the Pronase group or placebo group. The pretreatment solution was a mixed solution of 20000 U of Pronase and 60 m L sodium bicarbonate solution in the Pronase group, while an equal amount of sodium bicarbonate solution was administered to the placebo group. All operators, image evaluators and experimental recorders in EUS did not participate in the preparation and allocation of pretreatment solution. Two blinded investigators assessed the obscurity scores for the EUS images according to the size of artifacts(including ultrasound images of the gastric cavity and the gastric wall). Differences in imaging quality, the duration of examination and the usage of physiological saline during the examination process between the Pronase group and the control group were compared.RESULTS No differences existed in patient demographics between the two groups. For the gastric cavity, the Pronase group had significantly lower mean obscurity scores than the placebo group(1.0476 ± 0.77 vs 1.6129 ± 0.96, respectively, P = 0.000). The mean obscurity scores for the gastric mucosal surface were significantly lower in the Pronase group than the placebo group(1.2063 ± 0.90 vs 1.7581 ± 0.84, respectively, P = 0.001). The average EUS procedure duration for the Pronase group was 11.60 ± 3.32 min, which was significantly shorter than that of the placebo group(13.13 ± 3.81 min, P = 0.007). Less saline was used in the Pronase group than the placebo group, and the difference was significant(417.94 ± 121.38 m L vs 467.42 ± 104.52 mL, respectively, P = 0.016).CONCLUSION The group that had Pronase premedication prior to the EUS examination had clearer images than the placebo group. With Pronase premedication, the examination time was shorter, and the amount of saline used during the EUS examination was less. 展开更多
关键词 ARTIFACTS RANDOMIZED controlled trial ENDOSONOGRAPHY pronase STOMACH
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Efforts to increase image quality during endoscopy:The role of pronase 被引量:13
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作者 Gwang Ha Kim Yu Kyung Cho +2 位作者 Jae Myung Cha Sun-Young Lee Il-Kwun Chung 《World Journal of Gastrointestinal Endoscopy》 CAS 2016年第5期267-272,共6页
Clear visualization of the gastrointestinal mucosal surface is essential for thorough endoscopy. An unobstructed assessment can reduce the need for additional timeconsuming manipulations such as frequent washing and s... Clear visualization of the gastrointestinal mucosal surface is essential for thorough endoscopy. An unobstructed assessment can reduce the need for additional timeconsuming manipulations such as frequent washing and suction,which tend to prolong total procedure time. However,mucus,foam,and bubbles often hinder clear visibility during endoscopy. Premedication with pronase,a compound of mixed proteolytic enzymes,has been studied in order to improve mucosal visibility during endoscopy. Although its effects differ according to the location in the stomach,premedication with pronase 10 to 20 min before endoscopy significantly improves mucosal visibility without affecting the accuracy of Helicobacter pylori identification. The effects of pronase as premedication also extend to chromoendoscopy,narrow-band imaging,magnifying endoscopy,and endoscopic ultrasonography. In addition,endoscopic flushing with pronase during endoscopy may improve the quantity and the quality of a biopsy to some degree. Although improved mucosal visibility does not necessarily improve clinical outcomes,premedication with pronase may be helpful for increasing the detection rate of early cancers. 展开更多
关键词 ENDOSCOPY PREMEDICATION pronase
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基于固定化Pronase E酶与质谱的糖链结构分析方法 被引量:2
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作者 张曼丽 于子翔 +1 位作者 钱小红 应万涛 《分析试验室》 CAS CSCD 北大核心 2016年第1期1-6,共6页
位点特异性糖链结构的解析,是糖蛋白质结构分析面临的巨大挑战。Pronase E蛋白水解酶,能够降解糖蛋白或糖肽中大部分的氨基酸序列,而保留糖链与少量氨基酸的序列,与色谱、质谱分析等联用,可以实现糖链结构的鉴定,同时,保留的氨基酸序列... 位点特异性糖链结构的解析,是糖蛋白质结构分析面临的巨大挑战。Pronase E蛋白水解酶,能够降解糖蛋白或糖肽中大部分的氨基酸序列,而保留糖链与少量氨基酸的序列,与色谱、质谱分析等联用,可以实现糖链结构的鉴定,同时,保留的氨基酸序列可以辅助实现修饰位点的识别,两者结合,可以获取位点特异性糖链结构的信息。但Pronase E酶解的缺点是,酶解效率较低,常需要较高浓度的蛋白酶。本实验将Pronase E固定化在基质上,固定化后的Pronase E具备较高的局部浓度,从而实现目标糖蛋白的快速高效酶解。采用核糖核酸酶B作为标准糖蛋白,优化了Pronase E酶切的方案,包括酶切时蛋白与酶的用量比、酶切时间、固定化Pronase E酶的有效贮存时间等;同时优化选择了糖链的富集方法,并对于基质辅助激光解吸飞行时间质谱分析中,糖链适合的基质进行对比选择,从而获得更好的糖链谱图及更为丰富的糖链结构信息。 展开更多
关键词 糖蛋白 糖链结构 固定化pronase E 基质辅助激光解吸飞行时间质谱
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