【摘要】 糖基化蛋白对于生命体的生长发育,免疫调节,细胞识别粘附等具有重要意义,而异常的糖基化表达与风湿关节炎、肿瘤、阻塞性肺病等疾病密切相关.因此糖蛋白结构检测对于研究生命活动至关重要.由于在复杂样品中糖肽含量相对较少,加之非糖肽的离子抑制作用,使得糖肽的质谱检测有一定的挑战性.因此发展一种有效富集糖肽的方法是必要的.本实验中我们选用氧化铝对糖肽进行富集研究,并考察了影响氧化铝保留多肽的机理.我们利用氧化铝,从HRP酶解液中共获得16个糖肽,从IgG酶解液中共获得12个糖肽.与直接检测样品酶解液和经商品化材料Sepharose富集后再检测相比,检测到糖肽的个数增多.实验数据证明氧化铝富集糖肽具有较好的选择性和覆盖率.更多还原

【Abstract】 Protein glycosylation is significantly associated with cells cycle,immune regulation,cells recognition and cells adhesion.Aberrant glycosylation expression is involved in many diseases,such as rheumatoid arthritis,cancer,and chronic obstructive pulmonary disease.Thus,characterization of the protein glycosylation is very important to understand the life process.However,detection of glycopeptides is difficult by mass spectrometry because of the low concentration in complex sample and the suppressed signal by non-glycopeptides.Therefore,it is essential to explore an effective technique for glycopeptide enrichment.In this study,an alumina based-materials was used to selectively enrich glycopeptides.Firstly,we investigated the retention mechanism by changing the concentration of acetonitrile(ACN).With the decreased concentration of ACN,it was found that peptides were eluted according to their hydrophilicity.Moreover,most of the non-glycopeptides were eluted earlier than the glycopeptides in the high concentration of ACN fraction and the glycopeptides were found in the low concentration of ACN fraction.This result proved that hydrophilic interaction is one of the retention mechanisms for peptides retained by alumina.Secondly,we investigated the retention mechanism by changing the concentration of ammonium hydroxide.ACN were fixed at a high concentration and peptides were subsequently eluted with different concentration of ammonium hydroxide solution.At a low concentration of ammonium hydroxide solution,the peptides were no found.But at a high concentration of ammonium hydroxide solution,the glycopeptides and non-glycopeptides were eluted simultaneously.This proved that the ligand exchange is also one of the retention mechanisms,where the retention of the glycopeptides and non-glycopeptides showed no different under this mechanism.Based on the above mentioned results,the enrichment condition was optimized under the model of solid-phase extraction.High concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as loading buffer and low concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as elution buffer.The number of glycopeptides enriched by alumina were compared with glycopeptides before enrichment and enriched by Sepharose.In the tryptic HRP digest,7 glycopeptides were found before enrichment,16 glycopeptides were found by alumina and 14 glycopeptides were found by Sepharose.In the tryptic IgG digest,2 glycopeptides were found before enrichment,12 glycopeptides were found by alumina and 4 glycopeptides were found by Sepharose.In conclusion,alumina based-method used to enrich glycopeptides has high selection and wide coverage.更多还原