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Toward the Characterization of Mouse Protein Complexes

IRC Oct. 2005. Toward the Characterization of Mouse Protein Complexes. Jack Greenblatt University of Toronto. A Strategy for IDs of Stable Complexes and Weak Interactions. Tandem Affinity Purification. NO GEL!. SDS-PAGE. Trypsin Digestion. Active Protein For Assays. Gel Bands.

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Toward the Characterization of Mouse Protein Complexes

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  1. IRC Oct. 2005 Toward the Characterization of Mouse Protein Complexes Jack Greenblatt University of Toronto

  2. A Strategy for IDs of Stable Complexes and Weak Interactions Tandem Affinity Purification NO GEL! SDS-PAGE Trypsin Digestion Active Protein For Assays Gel Bands LCQ-Deca Ion Trap Mass Spectrometry Trypsin Digestion Identification of Stably and Weakly Associated Proteins MALDI-TOF Mass Spectrometry Identification of Stably Associated Proteins

  3. Purification of Tagged RNA Polymerase IIIdentification of Iwr1 no tag Rpb3 Rpb1 Rpb2 kDa 97 66 Rpb3-TAP 45 Rpb4 31 Rpb5 Ydl115c (Iwr1) 21 Rpb6 Iwr1 is an evolutionarily conserved transcription elongation factor.

  4. Iwr1 (Interacts with RNAPII) Proteins Identified from Rpb3-TAP by ION-TRAP Mass Spec

  5. 2005 RNA Polymerase I RNA Polymerase III RNA Polymerase II COMPASS Mediator Torpedo complex SWR-C CPF

  6. Data Format for Possible A-B Interaction score for identification of B by MALDI-ToF purification of A-TAP score for identification of B by LC/MS score for identification of A by MALDI-ToF purification of B-TAP score for identification of A by LC/MS

  7. TAP tags on all predicted yeast soluble proteins TAP purifications from 4004 different strains protein identifications from tandem MS and MALDI-ToF MS J48 decision tree naïve Bayes boosted stump decision tree stacking ROC curve (MIPS protein complexes as gold standard)

  8. 100% 90% 80% 70% 60% Sensitivity (TP Rate) 50% Toronto Data 40% Combined Others' Data Gavin 30% 20% 10% 0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1- specificity (FP Rate) ROC Curve Comparison of Yeast HTP Protein Interaction Studies IP/MS Ho Uetz Y2H Ito Haiyuan Yu and Mark Gerstein

  9. STRATEGY FOR MOUSE PROTEINS select mouse proteins use phage lambda red system to tag C-terminal of mouse protein on BAC in E. coli transform mouse ES cells with tagged BAC identify targeting event by PCR create tagged mice by aggregation with tetraploid embryos affinity purify tagged mouse protein from various tissues identify co-purifying proteins by tandem MS and MALDI-ToF MS

  10. attL exo bet gam cI857(cro-attR-bioA) P P Δ L R Tagging E. coli Proteins in vivo Using the λred Recombination System _________________________________________________________________________ Temperature sensitive cI repressor – inactive at 42°C λ Gam Exo/Bet – λ recombinase - two affinity tags KAN + STOP CODON CHROMOSOMAL ORF E. coli RecBCD - Exonuclease V

  11. Protein Complexes from E. coliComparison of TAP and SPA Systems SPA TAP InfC SufD SufD SufC SufC RpoD RpoD YacL SufB SufB YacL SPA TAP RpoB,C RpsA HepA SufB RpoD SufD InfC RpoA InfC RpsC,D SufC RpsG YacL RpsE,F,K,M,J NusG

  12. Essential Non Essential Interaction Network Containing the Most Highly Conserved, Soluble and Essential Proteins of E. coli RNA POLYMERASE ACP RIBOSOME ASSOCIATED IscS DNA POLYMERASE Butland et al. (2005) Nature 433: 531-537.

  13. B. Sem1-TAP No tag MR kDa Rpn1 97 Rpn2 66 Rpn3/Ubp6 Rpt1 Rpt2/Rpt3/Rpt5 Rpt4/ Rpn6 Rpn5/Rpn7 Rpt6 Rpn8/Rpn9 Rpn10/Rpn11 Rpn12 31 21 Rpn13 Sem1-TAP 14 The BRCA2 Interactor, DSS1, and its Yeast Homolog, Sem1, are Proteasome Subunits MR No tag DSS1-SPA kDa S1 (Rpn2) 97 S2 (Rpn1) S3 (Rpn3) 66 S4 (Rpt2) S5A (Rpn10) S6B (Rpt3) 45 S7 (Rpt1) S8 (Rpt6) S9 (Rpn6) S10A (Rpn7) S10B (Rpt4) S11 (Rpn9) 31 21

  14. Benoit Coulombe, 2005

  15. Tags Tested for Toxicity in Mouse ES Cells and Purification Efficiency Tag Purification Efficiency Toxic in ES cells? TAP (CBP + pA) + yes SPA (CBP + 3 X FLAG) + yes FA (3 X FLAG + pA) + somewhat HF (6 X HIS + 3 X FLAG) ++ no SF (SBP + 3 X FLAG) ++ nd

  16. Purification from E. coli of GFP with Various Tandem Affinity Purification Tags SPA SPA TAP FA HF FH SF FA HF SF Mr Mr 94 68 43 tagged GFP tagged GFP 30 21

  17. Purification of Tagged GFP from Mouse ES Cells, Embryos, and Various Tissues HF#1 ES cells muscle kidney testis brain heart lung liver embryos Mr FA HF#1 HF#6 HF#6 94 68 43 3FLAG-tagged GFP 6His-tagged GFP 30 TEV protease 21

  18. Organization of Cassette Used to Tag Mouse Proteins tag Lhom Rhom HF Pneo ATG Primers for wild-type allele Primers for homologous recombination FRT site for excision by FLPe Coding sequence of transcript

  19. KEY PEOPLE Greenblatt Lab Joyce Li Nevan Krogan Andrew Emili Gareth Butland Janet Rossant Marina Gertsenstein

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