Research Report 2010 - MDC

The principle standard SILAC (left) and pulsed SILAC (right). Instandard SILAC, cells are completely labeled by cultivating them ingrowth medium containing light (L) or heavy (H) stable (i.e. nonradioactive)amino acids. After several cell generations, all proteinshave uniformly incorporated the isotope label. Differentially labeldcells can be combined and analyzed together by mass spectrometry.The ratio of peak intensities reflects differences in proteinabundance between both samples. In pSILAC, cells are growing innormal (i.e. light) medium. Upon differential treatment, cells aretransferred to medium-heavy (M) of heavy (H) medium for pulselabelling. The ratio of heavy and medium-heavy peaks is a directmeasure of the differences in protein synthesis between bothexperimental conditions.of mRNAs. We are currently extending this analysis tomicroRNAs involved in carcinogenesis. (Olivia Ebner,Björn Schwanhäusser)Protein-protein interaction in neurodegenerativediseasesIdentifying interaction partners is the key to proteinfunction and can provide insight into disease mechanisms.Neurodegenerative diseases like Alzheimer’s arefrequently caused by accumulation of toxic proteinspecies in neuronal cells. We are using quantitativemass spectrometry and network analysis to analyzeprotein-protein interactions (PPIs) involved in diseasepathogenesis. A useful method to study PPIs is affinitypurification using a bait molecule. However, such pulldownassays suffer from the trade-off between sensitivityand specificity: While more stringent purificationconditions can remove some contaminating proteinsthey might also eliminate specific interaction partnerswith weak affinities and/or of low abundance.Quantitative proteomics solves this problem by comparingthe abundance of proteins identified in a pulldownexperiment with a suitable internal control. Thisallows the development of assays that can directly leadto understanding of biological systems. We are employingthis strategy to identify interaction partners of proteinsinvolved in neurodegeneration. Particularly, weare interested in identifying interactions affected bydisease-associated mutations. (Fabian Hosp, FlorianPaul)In vivo quantitative proteomics: The SILAC zooCell culture-based experiments cannot recapitulate allof the complex interactions among different cell typesand tissues that occur in vivo. Small animal modelssuch as worms, fruit flies and zebrafish are attractivealternatives that are extensively used in many areas ofbiomedical research, especially in genetics and developmentalbiology. While SILAC has enormouslyimproved quantitative proteomics in cultured cells, themethod was not yet used in small animal models. Weare currently extending this technology to three of themost important model organisms in biomedicalresearch: Caenorhabditis elegans, Drosophila melano gasterand Danio rerio. (Matthias Sury, Marieluise Kirchner)Selected publicationsSelbach M*, Paul FE, Brandt S, Guye P, Daumke O, Backert S, Dehio C,Mann M.* (2009) Host cell interactome of tyrosine-phosphorylatedbacterial proteins. Cell Host and Microbe 5, 397-403. (*sharedcorresponding authors)Schwanhäusser B, Gossen M, Dittmar G, Selbach M. (2009) Globalanalysis of cellular protein translation by pulsed SILAC. Proteomics 9,205-209.Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, Mann M. (2009) Apractical guide to the MaxQuant computational platform for SILACbasedquantitative proteomics. Nature Protocols 4, 698-705.Selbach M*, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, RajewskyN.* (2008) Widespread changes in protein synthesis induced bymicroRNAs. Nature 455, 58-63. (*shared corresponding authors)Selbach M, Mann M. (2006) Protein interaction screening by quantitativeimmunoprecipitation combined with knockdown (QUICK). NatureMethods 3, 981-983.Cardiovascular and Metabolic Disease Research 57