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

Abstract

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 Award	2017 Sept 1
Original language	English
Supervisor	Shu-Hui Chen (Supervisor)