Global Substrate Discovery For The Novel Deacetylase YcgC Of E. Coli Based By Quantitative Mass Spectrometry

Protein acetylation is a highly conserved post-translationalmodification from prokaryotes to eukaryotes. In the past few years, byhigh-resolution mass spectrometry combined with immunoprecipitationtechnology, tremendous of acetylated proteins and acetylation sites havebeen identified in bacteria, such as Acs and its609site acetylation. Instriking contrast, there is only one deacetylase CobB. Our lab develops anew method called “Clip-Chip” to identify novel deacetylases YcgC onan E.coli proteome chip and makes functional validations of enzymeYcgC and its substrates in vitro and in vivo. More importantly, YcgC isdifferent from CobB, so this study research for global substrate of YcgCand further analysis the substrate functions comparing with CobB.In this study, we identify the global substrates of YcgC and CobB bystable isotope labeling by amino acids in cell culture combined withhigh-resolution mass spectrometry and validate that in vitro and in vivoby bioinformatics analysis.Firstly, knockout strains by Red recombination technology andoverexpression strains of CobB and YcgC are constructed. Secondly,the appropriate glucose concentration and the induction time of YcgC areoptimized in order for the greatest difference acetylation between the YcgC overexpression and the wildtype strains.Thirdly, lysine is labeledby13C, we find a series of substrates of YcgC and CobB withhigh-resolution mass spectrometry and immunoprecipitation byanti-acetylation pan specific beads. Fourthly, we find a series of directlyinteracting protein of YcgC by purified YcgC on an E.coli proteome chip.Lastly, through analysing the results of mass spectrometry and E.coliproteome chip, we go bioinformatics analysis such as Motif Analysis,Functional Enrichment Analysis、 Protein-Protein Interaction NetworkAnalysis et.al, and select some candidates to validate in vitro bydeacetylation reaction. The main substrates of YcgC arerfaQ,trmD,rfbC,narH,frdA,nusB,hisJ,which have not the same with theknown CobB substrates, but the substrates panC,ydaL have a stronginteraction from the PPI microarray results.