Affinity Enrichment-based Proteomics Analysis Of Host Antiviral Response

Mass spectrometry(MS)-based quantitative proteomics has become an indispensable tool in life science research.In this study,we performed quantitative proteomics analysis utilizing affinity enrichment coupled with high performance mass spectrometry to analyze changes in host cell protein interactomes and protein ubiquitination upon virus infection.TBK1,STING,and MDA5 are three important players within the host antiviral innate immune response network.Systematic analysis of protein-protein interactions involving these three protein may shed new light on host antiviral innate immune responses.In Chapter two of this dissertation,we report the mapping of interactomes for TBK1,STING,and MDA5 by affinity enrichment MS in virally infected or uninfected THP-1 cells.To gain a more comprehensive view on host antiviral response induced by different virus,SeV and HSV,a RNA and a DNA virus respectively,are used.To maximally maintain a normal physiological state of host cells,endogenous expressed bait proteins were used.To ensure data quality,three biological replicates for each stimulus condition/bait combination were performed.In all,33 independent affinity enrichment mass spectrometry analysis were conducted resulting in the quantification of more than 2000 host proteins.Through stringent statistical analysis,13 proteins were identified as TBK1 interactors,31 were identified as STING interactors and 16 were identified MDA5 interactors.Our data indicated that only small changes occur among TBK1 and MDA5 interactors with or without virus stimulations while STING interacted with different proteins upon different viral infections,suggesting that STING activated a non-preexisting protein network upon viral infection.Functional analysis was performed on 17 interactors,and six were found to have functions in innate immune responses.We identified TTC4 as a TBK1 interactor and positive regulator of sendai virus-induced innate immunity.Ubiquitination is a common posttranslation modification which is widely involved in immune signaling pathways.In Chapter three of this dissertation,we describe a comparative proteomics analysis of protein ubiquitination in virally infected or uninfected THP-1 cells using triple SILAC labeling coupled with ubiquitination remnant immunoaffinity profiling followed by mass spectrometry analysis.We compared SeV infected or HSV infected cells to uninfected cells to analyze the ubiquitination change of host cell proteins.In all,more than 3600 ubiquitination sites of host cell proteins were identified and quantified.By analysing data normal distribution and selecting confidence interval of 95%,we identified 276 ubiquitination sites within 187 nonredondent host cell proteins as significantly changed upon viral infection.Our data indicated that there were more up-regulated ubiquitination sites than down-regulated sites upon virus infection.When comparing different types of virus stimulation,the up-regulated sites were mostly virus stimulation type specific but the down-regulated sites were often shared.Functional analysis was performed on eleven host cell proteins containing regulated ubiquitination sites,and eight were found to be likely to have functions in innate immune responses.