Phosphoproteome Analysis Of Pseudomonas Aeruginosa Stringent Response In Macrophages

As is known to all,eukaryotic posttranslational modifications play an important role incellular. Nowadays,serine, threonine and tyrosine phosphorylation in the regulation of cellularactivity research is hot. However, in prokaryotes, protein phosphorylation is widespread,two.Not only that, due to the many bacterial genome sequencing and bacterial proteomics, as well asmass spectrometry technology development, research found that, serine, threonine and tyrosinephosphorylation in many bacteria is common.In order to find the role of the change of protein in the bacterial response the conditions ofenvironmental stress, people research bacterial stress response. The ultimate goal is to explainthe stress response in the protein changes, analysis of their biochemical properties, to elucidatethe molecular mechanism for regulation of the stress response.This experiment put logarithmic phase Pseudomonas aeruginosa to mousemacrophages,then wash away bacterias not taken by macrophages.Lysis macrophages letPseudomonas aeruginosa out,Centrifugal collection it.Put Bacteria in lysis bufferultrasonication.Add DTT reduction and IAA alkylation.Add pancreatic enzymes cut priteins intopolypeptide segment.Polypeptide segment samples using high performance liquidchromatography reversed phase C18column to remove impurities.Then the samples using strongcation exchange column treatment.Then the desalted samples process by IMAC,using ferrum ioncolumn Enrich phosphopeptides.phosphorylation peptide mixture samples process by highperformance liquid chromatography mass spectrometry. Then search phosphorylation proteinand phosphorylation sites in database.This experiment identify24phosphorylated protein and57phosphorylation sites in controlgroup:31on serine,22on threonine,and nine on throsine.identify12phosphorylated protein and31phosphorylation sites in stress response group:7on serine,20on threonine,and four onthrosine.