Genome wide analysis of protein and mrna half lives reveals dynamic properties of mammalian gene expression
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1 Genome wide analysis of protein and mrna half lives reveals dynamic properties of mammalian gene expression Matthias Selbach Cell Signaling and Mass Spectrometry Max Delbrück Center for Molecular Medicine Berlin, Germany berlin.de/selbach
2 Dynamic regulation of gene expression Dynamic properties of gene expression depend on protein and mrna levels and half lives
3 Mathematical modeling of gene expression
4 Absolute mrna levels: next generation sequencing reads per kb of exon model per million mapped reads (RPKM) Mortazavi et al., N. Meth.,2008
5 Methods for absolute protein quantifaction Stable isotope based methods: AQUA peptides Gerber et al., PNAS, 2003 itraq Ross et al., MCP, 2004 QconCAT Beynon et al., N. Meth., 2005 absolute SILAC Hanke et al., JPR, 2008 Label free / computational methods: protein abundance index (PAI/emPAI) Ishihama et al, MCP, 2005 normalized spectral abundance factor (NSAF) Mosley et al., J.Prot., 2009 absolute protein expression measurements (APEX) Lu et al., Nat. Biotech, 2007 top3 peptide intensities (top3) Silva et al., MCP, 2006 Malstrom et al., Nature, 2009
6 Methods for absolute protein quantifaction Stable isotope based methods: AQUA peptides Gerber et al., PNAS, 2003 itraq Ross et al., MCP, 2004 QconCAT Beynon et al., N. Meth., 2005 absolute SILAC Hanke et al., JPR, 2008 Label free / computational methods: protein abundance index (PAI/emPAI) Ishihama et al, MCP, 2005 normalized spectral abundance factor (NSAF) Mosley et al., J.Prot., 2009 absolute protein expression measurements (APEX) Lu et al., Nat. Biotech, 2007 top3 peptide intensities (top3) Silva et al., MCP, 2006 Malstrom et al., Nature, 2009 intensity based absolute quantification (ibaq) Schwanhaeusser et al.,unpublished
7 Benchmarking different methods should be independent of protein sequence be accurate over several orders of magnitude should not be affected by overall sample complexity 48 recombinant human proteins over six orders of magnitude (UPS2, Sigma) + E. coli lysate (different amounts)
8 Benchmarking different methods
9 Benchmarking different methods
10 Cellular copy numbers of yeast and mouse (NIH3T3) proteins Yeast nucleosomes: 12 Mb genome / 150 bp = 80,000 nucleosomes ibaq: ~100,000 nucleosomes
11 Cellular copy numbers of yeast and mouse (NIH3T3) proteins mouse nucleosomes: 2.6 Gb haploid genome size x 2 / 150b = 3.5 x 10 7 nucleosomes ibaq: ~ 3 x 10 7 nucleosomes
12 Summary I: intensity based absolute quantification (ibaq) most accurate estimate of absolute protein abundance all available information is used (intensities of all peptides) spiked-in standard proteins minimize variability in sample handling very simple approach: universal standards can be used (AQUA, absolute SILAC) no complex machine learning procedures required (APEX) no need for three different MS methods (top3)
13 Large scale analysis of mrna and protein turnover Classical approach: inhibition of transcription / translation using drugs track mrna / protein decay over time Problem: drug treatment has severe side effects and ultimately kills the cells under study
14 Labeling with isotopes: Rudolf Schoenheimer Deuterium as an Indicator in the Study of Intermediary Metabolism, Schoenheimer and Rittenberg, J. Biol. Chem., 1935 In order successfully to label a physiological substance, it is essential that the chemical and physical properties of the labeled substance be so similar to the unlabeled one that the animal organism will not be able to differentiate between them. The chemist, on the other hand, must be able to distinguish and to estimate them in small quantities and at high dilutions. A possibility for such a label is the use of an isotope. Rudolf Schoenheimer,
15 Measuring protein dynamics using SILAC Relative changes in protein levels (Ong et al., 2002) Relative changes in protein production (Schwanhaeusser et al, 2009) protein turnover (Doherty et al., 2009)
16 psilac identification of mirna targets Nature, 2008
17 Metabolic pulse labeling of proteins and mrnas
18 Dynamic regulation of gene expression Dynamic properties of gene expression depend on protein and mrna levels and half lives
19 Dynamic properties of mammalian gene expression: mrna and protein levels and half-lives 4 Thiouridine
20 Metabolic pulse labeling of proteins
21 Summary parallel metabolic pulse labeling with SILAC and 4SU can be used to assess protein and mrna turnover at the same time (unperturbed system, no inhibitors) deep sequencing and mass spectrometry can quantify protein and mrna half lives and absolute cellular protein and mrna copy numbers (ibaq and RPKM) while mrna and protein levels are correlated, half lives are very different important consequences for dynamic properties of gene expression energy constraints posttranscriptional regulation (like RNA binding proteins) dynamic behavior
22 Thank you! Christian Sommer Dorothea Busse Jana Wolf Wei Chen Na Li Björn Schwanhäusser berlin.de/selbach
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