Mass Spectrometry Analysis Of Protein Post Translational Modification In Clinical And Basic Medical Research

Background:The rapid advancement in epi-proteomics technologies is providing more and morepowerful tools for detecting post-translational modification (PTM) of proteins. Wemanaged to set up an epi-proteomic analyzing platform of clinical and experimentalsamples with the latest mass-spectrometry technologies, with which we can find outunknown post translational modification of proteins by high throughput and highlysensitive LC-MS/MS analysis.Hepatic tumor marker HAAH is specifically over-expressed in multiple humancancer tissues, but its relationship with carcinogenesis is not clear; Amyotrophic lateralsclerosis (ALS) is a systemic neurodegenerative disease with pathogenic changes in theliver, which is marked by the formation of protein aggregates. The precise mechanism ofALS is not elucidated. Methods:Tissue and cell proteins were separated on a C18column with HPLC, followed byMS/MS analysis on LTQ Velos Pro or Q-STAR Elite mass-spectrometers. Posttranslational modifications of proteins were detected and functional studies wereperformed on different levels. Epi-proteomic platforms were set up on different basisincluding single protein, protein complex, whole cell lysate, whole tissue\biopsy and etc.Platform (I): Epi-proteomics of single protein. Samples include:1) Bands or spots cut from1D/2D SDS-PAGE gels (including target protein);2) Single protein from other resources.Platform (II): Epi-proteomics of protein complex. Samples include:1) Immunoprecipitated protein complex;2) Protein complex co-immunoprecipitated with other proteins;3) Protein complex from non-degenerate protein gels;4) Crude protein fractions isolated by other methods.Platform (III): Epi-proteomics of whole cell/tissue/biopsy. Samples include:1) Whole cell lysate from cell culture;2) Whole tissue lysate from animal and/or human tissues.Specific methods include:1) In-gel digestion;2) In-solution digestion;3) LC-MS/MS analysis on LTQ Velos Pro or Q-STAR Elite with differentdurations;4) Identify proteins of interest using SEQUEST;5) Identify known modifications of the proteins in the complex usingMASCOT/SEQUEST;6) Identify any possible modifications (known/unknown) on the proteins usingOPEN MODIFICATION. Results:Based on the epi-proteomic platforms, together with biochemical, molecularbiological and bio-informatic technologies, three different projects were carried out:1) PTM analysis of the downstream substrates of hepatic tumor marker HAAH andHumbug (cultured cells):We successfully cloned the genes of HAAH and Humbug. And the proteins wereexpressed in HEK293cells. Results indicate that the over-expression of HAAH andHumbug in could induce β-hydroxylase of different substrates on aspartyl/asparaginylresidues, among which, Heterogeneous nuclear ribonucleoprotein, Heat shock cognate71kDa protein and Heat shock70kDa protein1A/1B of HSP70family were closelyrelated to carcinogenesis.2) PTM analysis of spinal cord tissues of ALS patients (human tissue);Two-dimensional electrophoresis and mass-spec analysis revealed enormousdifferent modified proteins. Further analysis found out that GFAP (Glial fibrillary acidicprotein) was heavily acetylated in ALS spinal cord, and that larger fragments of GFAPwere increased in the in-soluble aggregates in ALS. Western blotting,immune-precipitation and mass-spec analysis revealed other different acetylated proteinsincluding Histones between ALS and non-ALS spinal cords.3) PTM analysis of SOD1G93A/+mouse tissues (model animal).GFAP acetylation is highly conserved in SOD1G93A/+mouse spinal cords comparedto ALS patients; Other acetylated proteins are highly different in ALS and non-ALSmouse spinal cords; Different Histones were acetylated in ALS and non-ALS mousespinal cords.Conclusions:1) The mechanism of carcinogenesis of the over-expression of HAAH and Humbugin tumor tissues probably lies in the hydroxylation of downstream proteins such asHeterogeneous nuclear ribonucleoprotein, Heat shock cognate71kDa protein and Heat shock70kDa protein1A/1B;2) Acetylation status of various proteins including GFAPand Histones is changed in ALS;3) and these changes are highly conserved in ALSmodel animal SOD1G93A/+, which may lead to breakthrough in the revelation of thepathogenesis of the disease.The thesis established epi-proteomic platforms of single protein, protein complexand protein profile. PTM studies of cultured cells, human tissue and model animal werecarried out. It is proved that based on the platforms set up here, enormous PTMinformation could be rapidly obtained from different resources. Together withbiochemical, molecular biological and bio-informatic technologies, valuableepi-proteomic information could be obtained and thus it could facilitate both clinical andbasic medical research.