Research Report 2010 - MDC

Structure of the GroupGroup LeaderDr. Gunnar DittmarGraduate StudentGünther KahlertTechnical AssistantsMijo StanicStart of group: January 2008Gunnar DittmarMass SpectrometryCellular signalingCells interact with their environment and react to differentstimuli and changes in the environment. Thesereactions can either be based on transcriptional activityor chemical modifications of existing proteins, so calledpost-translational modifications. In order to gaininsight into the regulation of these protein-interactionnetworks it is necessary to identify proteins, their modificationsand to measure the protein expression levelswithin a cell. This requires the identification of severalhundreds or thousands proteins and their quantificationin a complex mixture. In addition the informationhas to be collected in a time efficient way. All theserequirements are matched by modern mass spectrometryand result in the methods rise to be the defaultmethod for large-scale protein identification in life sciences.The core facility mass spectrometry offers a wide rangeof mass spectrometry methods for the identificationand quantification of proteins and peptides. Besides theidentification of proteins in gel slices, the mass spectrometrycore facility uses a number of proteomic techniquesin different collaborations with groups at theMDC. We are working closely with these groups to optimizethe methods for the different projects.Targeted proteomicsMonitoring the regulation of proteins under differentconditions can provide deeper insight in the regulatorynetwork, which underlies a signal transduction cascade.For many cascades the major players in thesepathways are already identified. Incorporating thisknowledge into a targeted strategy allows focusing tomonitor changes in the concentration of these players,avoiding the sequencing of unrelated, not regulatedproteins in the cell. A method that allows the selectionof a limited number of proteins is multiple reactionmonitoring (MRM). The technique has the advantage ofa high sensitivity combined with short run times on theliquid chromatography systems. This opens the possibilityof measuring large quantities of different samplesin a short time period and quantifying all componentsof the cascade.Several collaborations of the core facility within theMDC are now based on multiple reaction monitoringexperiments. The core facility has access to two differentmass-spectrometers capable for this type of experiments(Q-Trap 5500 and Q-Trap 4000).Non-targeted proteomic approachesMetabolic labeling of cells offers great advantages forthe quantification of proteins. The cells of interest aretherefore cultured in media, which contain isotopiclabeled (non radioactive) amino acids. The additionalmass of the amino acids can later be detected by massspectrometry. Comparison of two different samples ispossible by analyzing the sample on a high accuracymass spectrometer (LTQ-Orbitrap). The results of thesemeasurements are relative ratios for each proteindetected in the two different samples. This techniquesis currently used by the core facility for the quantificationof immuno-precipitations and large scale proteinidentifications.For samples, which cannot be cultured in media containingheavy amino acids, another technique is available.This technique relies on a highly reproducible liquidchromatography, since two different chromatographicruns have to match. Measurements of nonlabeledsamples are carried out on a Q-TOF premier as aMS E experiment.Identification of post-translational modificationsBesides the translational regulation of proteins, anotherlayer of regulation exists, which is mediated by post-222 Technology Platforms