Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks 英文参考文献.docVIP

QuantitativeAnalysisofProteinPhosphorylationsand InteractionsbyMulti-ColourIP-FCMasanInputfor KineticModellingofSignallingNetworks SumitDeswal1,2,AnnaK.Schulze3,ThomasHo¨fer3,WolfgangW.A.Schamel1,4,5* 1MaxPlanckInstituteofImmunobiologyandEpigenetics,andFacultyofBiology,BiologyIII,UniversityofFreiburg,Freiburg,Germany,2SpemannGraduateSchoolof BiologyandMedicine,Freiburg,Germany,3ResearchGroupModelingofBiologicalSystems,GermanCancerResearchCenterandBioQuantCenter,Heidelberg,Germany, 4BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany, 5Centre of Chronic Immunodeficiency (CCI), University Medical Center Freiburg,andUniversityofFreiburg,Freiburg,Germany Abstract Background: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transductioneventssuchasactivation-dependentphosphorylationofproteins,remainsonebottlenecktothissuccess. Methodology/PrincipalFindings:Weuse multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events tounrivalled precision. Inthis method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment ofthe kinase ZAP70 tothe TCR-CD3 and subsequent ZAP70 activation byphosphorylation inthemurine T-cell hybridoma andprimary murine Tcells. Counte