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Technology & Methods Seminar

Technology & Methods Seminar. “Blast and Other Methods of Probabilistic Sequence Comparison” Arcady Mushegian Bioinformatics Thursday, May 25, 1:00p.m. Classroom (1 st floor, Administration Building) Schedule with abstracts and previous presentation slides can be found on:

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Technology & Methods Seminar

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  1. Technology & Methods Seminar “Blast and Other Methods of Probabilistic Sequence Comparison” Arcady Mushegian Bioinformatics Thursday, May 25, 1:00p.m. Classroom (1st floor, Administration Building) Schedule with abstracts and previous presentation slides can be found on: K:\Weekly Seminar Schedule\Thursday -- Technology & Methods Information regarding previous seminars can be found at: http://research.stowers-institute.org/wiw/external/Seminars/index.htm

  2. . . Deeper into the MudPIT: Protein Lists and Beyond Laurence FLORENS LAF@stowers-institute.org

  3. Proteomics Rooms 351-357

  4. Over 2000 Samples Analyzed since Oct 2003

  5. Proteins MS/MS Spectrum High-Throughput Proteomics: MudPIT Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  6. Proteins MS/MS Spectrum Complex Protein Mixtures Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  7. Data Analysis Mass Spec Chromato Database Digestion What we need to know about your samples: • Details of prep: • Organism • Amino acid sequence(s) • Buffer • Concentration, Silver-stained gel, Western Blot • Protein content or PTMs Complex Protein Mixtures Types of samples we deal with: • Whole cell lysates • Membrane/Organelle preps • Co-IPed proteins • Affinity purified proteins • Any biochemically-sound protein mixture and appropriate negative controls Quantities: • low µg to 500µg Drop off: -80°C Freezer (Hallway Room 351)

  8. Complex Protein Mixtures - Troubleshooting • Sample form: • Small starting volume (<50ul) can be digested directly • Dried protein pellet after TCA-precipitation (PREFERRED) • Problems with “Sticky Stuff”: • Glycerol • High Detergent (e.g. 2% SDS) • DNA/RNA • Solutions: • Keep as low as possible or dialyze out • Methanol/Chloroform extraction • Benzonase w/o Benzonase w/ Benzonase • 13 IDed Proteins • 67 IDed Proteins

  9. MS/MS Spectrum Generating Peptides Proteins Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  10. Generating Peptides Proteomic Surveying: Endoproteinase Lys-C + Trypsin MKLSEVFEQE IDPVMQSLGY CCGRKLEFSP QTLCCYGKQL CTIPRDATYY SYQNRYHFCEKCFNEIQGES VSLGDDPSQP QTTINKEQFS KRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVC DGCLKKSART RKENKFSAKR LPSTRLGTFL DSMCRLELKL NSS

  11. Generating Peptides Proteomic Surveying: Endoproteinase Lys-C + Trypsin MKLSEVFEQE IDPVMQSLGY CCGRKLEFSP QTLCCYGKQL CTIPRDATYY SYQNRYHFCEKCFNEIQGES VSLGDDPSQP QTTINKEQFS KRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVC DGCLKKSART RKENKFSAKR LPSTRLGTFL DSMCRLELKL NSS PTMs:High Sequence Coverage MSQAIAEKQP SQEVKMEAKMEVDQPEPADT QPEDISESKV EDCKMESTET EERSTELKTE IKEEEDQPST SATQSSPAPG QSKKKIFKPE ELRQALMPTL EALYRQDPES LPFRQPVDPQ LLGIPDYFDI VKSPMDLSTI KRKLDTGQYQ EPWQYVDDIW LMFNNAWLYNRKTSRVYKYC

  12. Generating Peptides Proteomic Surveying: Endoproteinase Lys-C + Trypsin MKLSEVFEQE IDPVMQSLGY CCGRKLEFSP QTLCCYGKQL CTIPRDATYY SYQNRYHFCEKCFNEIQGES VSLGDDPSQP QTTINKEQFS KRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVC DGCLKKSART RKENKFSAKR LPSTRLGTFL DSMCRLELKL NSS PTMs:High Sequence Coverage MSQAIAEKQP SQEVKMEAKMEVDQPEPADT QPEDISESKV EDCKMESTET EERSTELKTE IKEEEDQPST SATQSSPAPG QSKKKIFKPE ELRQALMPTL EALYRQDPES LPFRQPVDPQ LLGIPDYFDI VKSPMDLSTI KRKLDTGQYQ EPWQYVDDIW LMFNNAWLYNRKTSRVYKYC Post-Translational Modifications: Triple/Multiple Digest • Sample is split into x aliquots • Digest using x different proteases • Analyze samples individually • Interpret spectra using SEQUEST Trypsin Elastase Subtilisin MudPIT MudPIT MudPIT • MacCoss et al. (2002) Proc Natl Acad Sci U S A. 99:7900-7905

  13. Generating Peptides: DmSNS (1479 AA, 162kDa) 86.6% 5 Ti + 2 Es + 1 PK SP Extracellular … • Maggie Chen, Kiran Kocherlakota, Jeff McDermott

  14. Cytodomain • Maggie Chen, Kiran Kocherlakota, Jeff McDermott

  15. Generating Peptides - Troubleshooting • No tryptic peptides are obtained from particular protein • Overdigestion with non-specific proteases • No positively charged amino acids in peptides

  16. MS/MS Spectrum Multidimensional Chromatography Proteins Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  17. Multidimensional Chromatography Small digest volumes (<200µl): 3-phase 100µm Fused Silica Large digest volumes (>200µl): split-3-phase 250µm Fused Silica 100µm FS 100µm FS 250µm FS RP - C18 RP - C18 waste Filtered Union with 2µm Frit SCX 250µm FS 250µm FS RP - C18 SCX RP - C18 Peptides Peptides

  18. Multidimensional Chromatography • Cycle 1 – Reverse Phase Gradient • Cycle 2_Step 1 – Salt Pulse • Cycle 2_Step 2 – Reverse Phase Gradient • Cycle 3_Step 1 – Increase Salt Pulse • Cycle 3_Step 2 – Reverse Phase Gradient H2O MeCN NH4OAc HPLC 2.4kV Number of Cycles depends on Sample Complexity

  19. MS/MS Spectrum Tandem Mass Spectrometry Proteins Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  20. Tandem Mass Spectrometry Deca-XP vs LTQ (3D vs Linear Ion Trap) • Hardware improvements for LTQ: • Trapping efficiency (no rf field in ion injection axis), • Ion capacity (linear configuration of mass analyzer -> larger volume), • ~2x Detection efficiency (radial ejection of ions + 2 detectors) • ~3x Ion ejection rate (while maintaining same resolution) • Should significantly improve the number of detected peptides/proteins • Blackler et al. (2006) Anal. Chem.78:1337-13344

  21. Tandem Mass Spectrometry Deca-XP vs LTQ ~10µg FLAG-tagged Mediator Prep • ~2.5x more Protein IDs • More Proteins that matter: not necessarily • More spectra per protein (Quantitation, PTMs)

  22. Tandem Mass Spectrometry LTQ Varying amounts ofPfs25 (185AA, 20kDa) Constant HsMediator (~10µg Total) XP Varying amounts of SpRunt1 (535AA, 59kDa) Constant HsMediator (~20µg Total) • Both machines: Detection limit is ~0.05% of Total Protein Quantity (w/w) • LTQ: more peptides/spectra detected for same quantities, i.e. greater confidence

  23. Tandem Mass Spectrometry • The increase in number of spectra obtained from the LTQ results in: • much larger files (x10) • significantly increase in computational overhead (Storage and Search Time) • Blackler et al. (2006) Anal. Chem.78:1337-13344

  24. Computational Overhead • Dan Thomasset

  25. Computational Overhead • Dan Thomasset

  26. Instrumentation Priorities • XP • On-going Protein Identification Projects LTQ Quantitation (isotopic labeling) PTMs Low Abundance Proteins New Protein Identification Projects

  27. MS/MS Spectrum Matching MS/MS Spectra to Peptides Proteins Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  28. Shuffled Sequences: • Used to estimate False Discovery Rates (FDR) Matching MS/MS Spectra to Peptides Search engine: SEQUEST® Protein Sequence Databases: • Need to be as comprehensive as possible (whole genomes) • Need to include “custom” sequences • Updates

  29. MS/MS Spectrum Data Analysis Proteins Peptide Mixture Cells Tissues Embryos Lysis Purification Digestion Tandem Mass Spectrometry 2D Chromatography Protein List Data Analysis Database Matching

  30. Filtering & Assembling Data: DTASelect SQTs • Through P: drive • http://bioinfo/proteomics/ PARSE ASSEMBLE DTASelect.html (DTASelect-filter.txt) DTASelect FILTER High-stringencyFiltering Criteria to limit FDR: • Cross-correlation score (XCorr) • DeltaCN • Peptide Length • Peptide Ends • Tabb et al. (2002) J Proteome Res1: 21-26

  31. Comparing Protein Lists: CONTRAST n Protein Lists CONTRAST COMPARE Contrast.html (Contrast.txt) MERGE Text files MSAccess • Tabb et al. (2002) J Proteome Res1: 21-26

  32. Comparing Protein Lists: contrast-report n Protein Lists • Mike Coleman contrast-report COMPARE report.xls • Dan He

  33. Comparing Protein Lists: contrast-report n Protein Lists • Mike Coleman contrast-report COMPARE report.xls • Dan He

  34. Relative Abundance: NSAF • Quantitative Information from MudPIT dataset? • Sequence Coverage: high for small proteins / low for large proteins • Spectral Count: large proteins contribute more peptides/spectra Normalized Spectral Abundance Factor • Values between 0 and 1 • Best approximation of protein levels in a sample • Allows comparisons across multiple runs and across different instruments

  35. Relative Abundance: NSAF • Mingan Shi • Erika Geisbrecht

  36. PTM Analysis • Glycosylation: • N-linked: PNGFase, which leaves modified N (Dmass= +1 Da) • O-linked: beta-eliminate O-glycosylations with NH4OH, which leaves modified S and T (Dmass= -1 Da)

  37. PTM Analysis Flowchart

  38. Mike Coleman ptm-report

  39. Mike Coleman contrast-ptm

  40. Time-line PTMs Searches LC/LC-MS/MS PTMs Results CONTRAST DTASelect Digestion Search 1 week 1 Month 2 weeks 3-6 Months

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