【作者】 郭欣；

【导师】 董铭铭； 叶明亮；

【作者基本信息】 大连理工大学 ， 生物与医药， 2024， 硕士

【摘要】 蛋白核心岩藻糖基化(CF)修饰的异常在癌症的发生、进展和免疫抑制中起着关键作用,核心岩藻糖基化的蛋白质是一类重要的疾病治疗靶点和肿瘤标志物。受限于其低丰度及复杂的糖链结构,其高灵敏表征仍存在巨大挑战。同时,由于完整糖肽的质谱响应通常受糖链组成的影响,导致在完整糖肽层次进行核心岩藻糖基化位点占有率的准确测定存在困难。为了克服这些挑战,我们发展了一种高效的N-聚糖截短策略,通过酶促作用将含有数百种不同N-糖链的完整糖肽转化为两种截短形式(截短糖链的HexNAc型糖肽及截短糖链的CF型糖肽),极大程度上降低了N-糖基化的异质性,同时更有利于质谱对于肽段骨架的碎裂。此外,聚糖截短策略也促进了质谱数据解析过程,截短糖链的N-糖肽可通过质谱非依赖无标记定量策略分析,实现了核心岩藻糖基化肽段的高灵敏、高重现定性定量分析,且可实现核心岩藻糖基化位点占有率的精准测定。研究内容及结果如下:(1)内切糖苷酶的表达及纯化:通过原核系统成功表达及纯化出Endo M、Endo F3两种具有良好酶切互补性的内切糖苷酶,通过IgG、Fetuin两种标准蛋白完成酶活力的测试,采用健康人血清完整糖肽样品完成酶切效率系统性评估。采用聚糖截短策略后样品中完整糖肽类鉴定数目比处理前减少了98.0%,剩余完整糖肽的强度也比相同序列的截短糖链的N-糖肽低25倍,说明使用具有互补特异性的内糖苷酶可以实现对N-聚糖的有效切割。(2)策略可行性分析:采用健康人血清完整糖肽样品进行聚糖截短策略的可行性评估,与未截短策略(即N-完整糖肽层次)相比,使用聚糖截短策略后具有核心岩藻糖(CF)位点的肽段序列的鉴定量增加了140%。同时对比了数据依赖性采集(DDA)和数据非依赖性采集(DIA)模式对截短型N-糖肽的鉴定性能。与DDA相比,DIA具有鉴定灵敏度高、覆盖度高、缺失值少、定量重现性更好等优点。(3)应用于乳腺癌肿瘤细胞核心岩藻糖基化的差异表达分析:从MDA-MB-231和MCF7中共鉴定到1615个独特的糖基化位点且单次LC-MS/MS分析可鉴定约900个CF位点。通过对两种细胞系CF位点丰度和占有率的差异分析,发现201个细胞表面药物靶点在乳腺癌亚型之间具有不同的糖基化模式。发现了PDL1仅在MDA-MB-231细胞中表达且包含两个高度核心岩藻糖基化的位点;NOTCH3仅在MCF7细胞中表达且包含两个高度核心岩藻糖基化的位点;此外在三阴性乳腺癌细胞MDA-MB-231中,ERBB2的低表达使其靶向治疗效果不佳,本研究发现ERBB1蛋白中存在7个高度上调的糖基化位点,这些位点为TNBC的治疗提供新思路。综上,该策略可实现CF丰度及其位点占有率的同时分析,其在癌症机制研究、新型疾病标志物发现、药物干预靶点鉴定等方面有广泛的应用前景。更多还原

【Abstract】 Dysregulation of protein core-fucosylation(CF)plays pivotal roles in the onset,progression and immunosuppression of cancer.Protein core-fucosylation are an important class of disease treatment targets and tumor markers.Due to their low abundance and complex glycan structures,their high-sensitivity characterization still poses a significant challenge.Additionally,due to the glycan composition influencing the mass spectrometric response of intact glycopeptides,accurate determination of CF occupancy ratio at the intact glycopeptide level is difficult.To overcome these challenges,we developed an efficient N-glycan truncation strategy,which efficiently convert intact glycopeptides with hundreds of different N-glycans into two truncated forms,i.e.a monosaccharide HexNAc and a disaccharide HexNAc+core Fuc.This approach significantly reduces the heterogeneity of N-glycosylation,thereby facilitating mass spectrometric fragmentation of the peptide backbone.Furthermore,the glycan truncation strategy enhances the MS data interpretation process.Truncated N-glycopeptides can be analyzed using a label-free of data-independent analysis(DIA)strategy,achieving high sensitivity and high reproducibility in both qualitative and quantitative analysis of corefucosylated peptides,and enabling accurate measurement of the CF occupancy ratio.The research content and results are as follows:(1)Expression and purification of endoglycosidases: Endo M and Endo F3,two endoglycosidases with complementary specificity on substrate glycans,were successfully expressed and purified through a prokaryotic system.The enzymatic activity was initially explored using two standard proteins,IgG and Fetuin.A systematic evaluation of enzymatic efficiency was completed using intact glycopeptides from healthy human serum.After applying the glycan truncation strategy,it can be observed that the number of intact glycopeptides in the system has decreased by 98.0% compared to non-treatment.The intensity of the remaining intact glycopeptides is also 25 times lower than that of the truncated glycopeptides with the same peptide sequence.This validates that endoglycosidases with complementary specificity can effectively cleave N-glycans.(2)Feasibility analysis of the strategy: We use human serum sample collected from healthy people to test the feasibility of this strategy.The truncated strategy identified 140% more peptide sequences with CF sites compared to the un-truncated strategy(i.e.the intact glycopeptide).At the same time,the identification performance of truncated N-glycopeptides under two mass spectrometry modes,DDA and DIA,was compared.Compared with DDA,DIA has advantages such as higher identification sensitivity,higher identification quantity,higher coverage,fewer missing values,and better quantitative reproducibility.(3)Differential expression analysis of core-fucosylation in breast cancer cell lines: i.e.MDA-MB-231 and MCF7,yields a total of 1615 unique glycosylation sites and about 900 CF sites from one single LC-MS/MS analysis.Differential analysis unraveled the distinct glycosylation pattern for over 201 cell surface drug targets between breast cancer subtypes.It was found that PDL1 is only expressed in MDA-MB-231 cells and contains two highly corefucosylated sites;NOTCH3 is only expressed in MCF7 cells and contains two highly corefucosylated sites;in addition,in triple-negative breast cancer cells MDA-MB-231,the low expression of ERBB2 results in poor targeted therapy effects.This study found that there are 7highly up-regulated glycosylation sites in the ERBB1 protein.These sites provide new insights for the treatment of TNBC.In summary,this strategy can simultaneously analyze the abundance of CF and site occupancy ratio.It has broad application prospects in cancer mechanism research,discovery of new disease markers,identification of drug intervention targets.更多还原