中文版 | English
题名

基于两性整体柱的蛋白质组学样品前处理技术的开发

其他题名
DEVELOPMENT ZWITTER-IONIC MONOLITH-BASED PROTEOMIC SAMPLE PREPARATION TECHNOLOGY
姓名
姓名拼音
SU Yiran
学号
11930529
学位类型
硕士
学位专业
0703 化学
学科门类/专业学位类别
07 理学
导师
田瑞军
导师单位
化学系
论文答辩日期
2022-05-13
论文提交日期
2022-06-20
学位授予单位
南方科技大学
学位授予地点
深圳
摘要
  基于质谱的蛋白质组学为疾病发生发展、生物学规律和机制的探究提 供了强有力的依据,而蛋白质组学样品前处理技术是限制蛋白质鉴定深度 和鉴定通量的关键步骤。本论文开发了基于两性整体柱的样品前处理技术。
在热引发/光引发下聚合得到的两性整体柱通透性好,与蛋白质结合能力强, 可以实现蛋白质富集、还原、烷基化和酶解的全部过程。毛细管柱式两性 整体柱(ZIM-Column)主要用于纳克级样品的高灵敏度蛋白质组学分析, 其柱体积小,毛细管柱内壁修饰亲水性基团,减少了样品前处理过程中的 非特异性吸附。当两性整体柱和多肽除盐所用的 C18 填料存在于同一根毛
细管柱时,10 纳克 293T 细胞裂解液可以在 80 分钟的质谱分析中鉴定超过 4500 种蛋白质。移液枪头式两性整体柱(ZIM-Tip)则主要用于微克级样 品的高通量蛋白质组学分析,所有的样品前处理流程可在 2 小时内完成。
ZIM-Tip Evosep One 液相色谱系统联用时,1 微克 293T 细胞裂解液可 以在 1 小时的质谱分析中稳定鉴定超过 4000 种蛋白质。更为重要的是, 该技术具有很好的日内重复性和日间重现性,其变异系数的中位值分别小 于 20%30%Pearson 相关系数的平均值均大于 0.9。总之,基于两性整体柱的样品前处理技术为需要高灵敏度或高通量的临床蛋白质组学研究提
供了一种潜在的解决方案。
其他摘要
  Mass spectrometry (MS)-based proteomics provides a powerful basis for the study of disease development, biological characteristic and regulatory mechanism, but proteomic sample preparation limits the depth of protein identification and throughput. In the thesis, we prepared zwitter-ionic monolith- based one-pot proteomics processor, based on thermal/photo-initiated polymerization, with proper permeability and satisfying protein binding capacity. Proteins can be preconcentrated, reduced, alkylated and digested on the zwitter-ionic monolith. The zwitter-ionic monolith-based capillary column
(ZIM-Column) is mainly used for high-sensitivity proteome profiling of ng- level samples. ZIM-Column has limited column bed volume and hydrophilic modified inner wall which can reduce the non-specific adsorption during sample preparation process. For an 80-minute single-shot MS analysis, more than 4500 proteins were identified from 10 nanograms of 293T cell lysate when the zwitter-ionic monolith and the C18 matrix used for peptide desalting were present in the same capillary column. The zwitter-ionic monolith-based spintip column (ZIM-Tip) is mainly used for high-throughput proteome profiling of μg- level samples, which seamlessly match the Evosep One liquid chromatography system. The sample preparation workflow can be completed within 2 hours. For a 1-hour single-shot MS analysis, more than 4000 proteins were consistently identified from 1 microgram of 293T cell lysate. Excellent intra- and inter-day reproducibility in quantification were demonstrated with median coefficient of variation values less than 20% and 30%. The average Pearson correlation
coefficients of each two sets of samples are 0.934 and 0.901, respectively.
Zwitter-ionic monolith-based one-pot proteomics processor will help clinical studies demanding large sample amounts or in-depth proteome coverage.
关键词
其他关键词
语种
中文
培养类别
独立培养
入学年份
2019
学位授予年份
2022-06
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化学系
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苏怡然. 基于两性整体柱的蛋白质组学样品前处理技术的开发[D]. 深圳. 南方科技大学,2022.
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