基于数字液滴微流控芯片的高通量蛋白质组学分析平台构建

HIGH THROUGHPUT PROTEOMIC S PLATFORM BASED ON DI GITAL MICROFLUIDIC CHIP

孙秀杰

11649072

硕士

化学

田瑞军

2018

2018.7.6

哈尔滨工业大学

深圳

蛋白质组学是对蛋白质的结构和功能的大规模研究。目前，基于质谱的方法已经被广泛地应用于蛋白质组学中。蛋白质组学分析的基本流程是样品的前处理、蛋白分离、质谱分析鉴定以及生物信息学的分析。蛋白质组学样品分析前需要进行的处理有：细胞裂解、蛋白质预富集、蛋白质二硫键的打开与保护、缓冲液的交换、烷基化、蛋白质酶解和多肽组分的除盐等。在目前的蛋白质组学分析策略中，复杂的样品前处理步骤大大地降低了样品处理的通量，同时增加了样品的损失并降低了相应反应的灵敏度和反应效率。本工作运用数字微流控（Digital Microfluidics，DMF）技术来发展具有集成化和高通量特点的蛋白质组学样品前处理平台。预期以液滴为微反应器的样品单元将蛋白质前处理步骤所用溶液变换成液滴的微液滴的模式；通过液滴之间的融合和分离来完成蛋白质前处理过程，以期实现蛋白组学样品处理的微型化和集成化。本工作并将带有离子交换基团的磁珠基团用于微流控芯片蛋白质反应器，并对磁珠的类型进行了前期的探索，主要为将前期已发展的离心式集成化、微型化蛋白质组学分析技术SISPROT（Simple and Integrated Spintip-Based Proteomics Technology）中的SCX填料用SCX和SAX混合填料来替换，系统地考察了蛋白质在SCX/SAX混合填料上的保留行为和酶解步骤中因pH变化所引起的蛋白质损失。另外，我们还就DMF技术应用于分析多肽的高通量人工合成进行了探索性研究。

Proteomics is a large scale study of protein structure and function, including the interaction between protein and protein, protein expression leve l, post translational modification, etc. At present, proteomics based on mass spectrometry has been widely used.The basic workflows of proteomics analysis using mass spectrometry as a means of detection are sample preparation, protein separation, mass spe ctrometry analysis and bioinformatics analysis Before the analysis of proteomics sample, many steps preparation need to be accomplished, like cell lysis, protein preconcentration, the opening and protection of protein disulfide bonds, protein enzymatic hyd rolysis and salt removal of peptides. In the present proteomics research strategies, complicated sample preparation step s are greatly reduced the flux of sample processing, which increase the loss of the sample the reaction efficiency and reduce the corre sponding at the same time This work use s d igital m icrofluidic s (DMF) technology to develop a n integrated and high throughput proteomics sample preparation platforms It is expected that the droplets us ed as microreactor sample unit complete the protein pr eparation process by fusing and splitting in order to realize the miniaturization and in tegration of proteomics sample processing In this work, magnetic beads with io n exchange groups were used in digital m icrofluidic protein reactors, and a previous exp loration of the type of magnetic beads was carried out, mainly to develop an integrated technology for preparing proteomics samples based on SCX/SAX mixed beads. And this work systematically investigates the retention behavior of the protein and the protei n loss due to the pH change during the enzymatic hydrolysis step on the SCX/SAX mixed beads. Furthermore, we also conducted exploratory studies on the high throughput artificial synthesis of DMF technology for proteins.

蛋白质组学样品前处理 SISPROT 离子交换 数字液滴微流控 蛋白吸附 多肽合成

proteomics sample preparation SISPROT ion exchange digital microfluidics prevention against protein biofouling peptide synthesis

中文

联合培养

南方科技大学

孙秀杰. 基于数字液滴微流控芯片的高通量蛋白质组学分析平台构建[D]. 深圳. 哈尔滨工业大学,2018.