Research Report 2010 - MDC

Structure of the GroupMatthias SelbachGroup LeaderDr. Matthias SelbachScientistsDr. Marieluise KirchnerDr. Matthias SuryGraduate StudentsOlivia EbnerFabian HospFlorian PaulBjörn SchwanhäusserTechnical AssistantsChristian SommerSecretariatSonja GieringPetra HainkCell Signaling and Mass SpectrometryProteins are the chief actors in almost every biological process. While we know a lot aboutthe function of individual proteins there is little information about the system as a whole.Recent developments in mass spectrometry have dramatically improved the analytical powerof this technology. We are using mass spectrometry-based quantitative proteomics toinvestigate cellular signaling at the protein level on a global scale. Main areas of research arepost transcriptional regulation of gene expression by microRNAs, protein-protein interaction inthe context of neurodegenerative diseases and in vivo quantitative proteomics.Recently developed quantitative methods make it possibleto obtain precise functional information and tomonitor temporal changes in the proteome by massspectrometry. In one approach, named SILAC (for stableisotopelabelling with amino acids in cell culture), cellsare differentially labeled by cultivating them in thepresence of either normal or a heavy isotope–sub -stituted amino acid, such as 13 C-labeled lysine (Figure,left panel). Due to their mass difference, pairs of chemicallyidentical peptides of different stable-isotope compositioncan be distinguished in a mass spectrometer.The ratio of intensities for such peptide pairs accuratelyreflects the abundance ratio for the corresponding proteins.Quantitative proteomics with SILAC has emergedas a very powerful approach to investigate signalingprocesses. We are using this technology as our centraltool to address challenging questions in cell signaling.pSILAC and microRNAsRegulation of gene expression occurs at all stages frommRNA transcription to protein synthesis. It is now clearthat translation itself is a regulated process with a centralrole in cellular physiology and a growing catalogueof human diseases. A prominent example aremicroRNAs: These small non-coding RNAs repress targetgenes by inhibiting translation or by inducingdegradation of mRNAs. In mammals, microRNAs arepredicted to control the activity of ~30% of all proteincodinggenes. They have been shown to be involved inregulation of almost every cellular process investigatedso far. In order to understand microRNA function it iscrucial to investigate how they regulate protein pro -duction.We developed pulsed SILAC (pSILAC) as a novel methodto directly compare protein translation rates betweentwo samples (Figure, right panel). Cells are first cultivatedin standard growth medium with the normal light(L) amino acids. Concomitantly with differential treatment,cells are transferred to culture medium containingheavy (H) or medium-heavy (M) amino acids. Allnewly synthesized proteins will be made in the H or Mform, respectively. Subsequently, both samples are combinedand analyzed together. The abundance ratio of Hversus M peptides reflects differences in translation ofthe corresponding proteins integrated over the pulselabelling incubation time. We employed pSILAC tomeasure changes in synthesis of several thousand proteinsafter misexpression of microRNAs (collaborationwith the group of Nikolaus Rajewsky). Bioinformaticanalysis of the data showed that a single microRNA candirectly repress hundreds of targets and that thisrepression is typically rather mild. By comparing thepSILAC and the microarray data we were able to showthat microRNAs directly repress translation of hundreds56 Cardiovascular and Metabolic Disease Research