Fabrication of On-Line Nano-Liquid Chromatography-Mass Spectrometry for Top-down Proteomics and Application for the detection of Modified Hemoglobin in Red Blood Cells

  • 賴 威彣

Student thesis: Master's Thesis


Estrogen and its metabolites have been reported to form covalent bonds with proteins causing changes of protein functions which are related to diseases Although conventional bottom-up proteomics technique can be used to identify modified proteins it is still rather time-consuming and inconvenient for biomarker detection In this research we tried to develop a top-down method for identifying hemoglobins modified by catechol estrogens For this purpose we develop a packing method for fabricating nano columns and apply the self-packed column for top down analysis of modified hemoglobin in red blood cells using LC-MS/MS and Prosight PTM 2 0 for proteomic search The bottom-up approach was further applied to confirm the modification sites identified by the top-down approach Sample used in this research is the lysed red blood cells which were co-incubated with 4-hydroxyestradiol in order to generate modified hemoglobin Two packing materials were attempted for packing Our data indicated that the self-packed nano-column had reasonable flow resistance for 5 μm reversed phase C18 particles but was relatively loose for 20 μm reversed phase particles But our results showed the 20 um column can still separate hemoglobin ? and β chain as well as 4-hydroxyestradiol modified hemoglobin ? and β chain at a relatively faster speed In the MS1 spectrum can observe 4-hydroxyestradiol modification on hemoglobin ? and β chain When the raw data acquired by using the 20 um colume were analyzed with Prosight PTM 2 0 the modification site could not be confirmed due to few fragments by collision induced dissociation Moreover the modification site identified by the top-down approach was able to be confirmed by the bottom-up approach The modified hemoglobin β chain however could not be identified by the top-down approach but was identified by the bottom-up approach using chymotrypsin digestion Our data provide evidences that top-down method may be developed for biomarker detection of estrogenized hemoglobin
Date of Award2017 Sept 1
Original languageEnglish
SupervisorShu-Hui Chen (Supervisor)

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