报告人：Myeong Hee Moon 教授

报告题目：Field-flow Fractionation with Mass Spectrometry for Proteomic & Lipidomic Analysis

报告时间：2019年10月23日，上午10:00

报告地点：化学楼一楼会议室

报告人简介：

Field-flow Fractionation with Mass Spectrometry for Proteomic & Lipidomic Analysis Myeong Hee Moon Dept of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul, 03722, Korea Field-flow fractionation (FFF) is a separation method capable of separating particles and biological macromolecules like proteins, DNA, cells, and etc by sizes in an empty channel space without packing materials. Flow FFF (FlFFF), a variant of FFF family utilizing cross flow as driving force of separation in addition to migration flow, provides a gentle but high speed size separation of biological macromolecules such as proteins, cells, DNA, subcellular species in a biological buffer solution. Especially for the biomaterials, high performance separation/isolation methods are crucial to reduce the sample complexity even though sophisticated use of mass spectrometry (MS) is available. This presentation will show recent advances of FlFFF for the size separation of biomolecular complexes such as lipoproteins, exosomes, subcellular species, and metalloproteins and followed by proteomic and lipodomic analysis using nanoflow liquid chromatography tandem mass spectrometry (nLC-ESI-MS/MS). Lipoproteins in blood are globular complexes containing phospholipids, cholesterols, triacylglycerols, and etc. Among lipoproteins, low density lipoprotein (LDL) is known to be strongly related to coronary artery disease (CAD). In bottom-up approach, different lipoproteins can be size-sorted by FlFFF and lipids including oxidized PLs extracted from each collected lipoprotein were analyzed by nLC-MS/MS with structural determination. Covered topics include the application of FlFFF for a high speed, semi-preparative scale, biocompatible size sorting of subcellular organelle species from homogenate mixtures of HEK 293T cells using FlFFF system will be introduced with confirmation of collected fractions containing nuclei, lysosomes, mitochondria, and peroxisomes using SEM, Western blot, and proteomic analysis with nLC-MS/MS. Applications to exosomes, extracellular vesicles secreted from human urine sample with prostate cancer, will be discussed. Lastly, it will be introduced with a result from the collaborative research with Chinese Academy of Science. A fully automated on-line method was developed to carry out proteolysis and glycopeptide enrichment in sequence for nLC-ESI-MS/MS for glycoprotein analysis by implementing two serial thermo-responsive porous polymer membrane reactors (TPPMRs), in which the TPPM could be immobilized either with trypsin for proteolysis or with lectins for glycopeptide enrichment. With TPPMRs, the entire pretreatment procedure can be performed on-line in about an hour and the one step injection of proteome sample can be analyzed for glycopeptides.