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Proteomics

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Proteomics

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Proteomics

  1. 1. Proteomics Protein identification using mass spectrometry www.draptis.eu/proteomics.ppt
  2. 2. Introduction <ul><li>Proteomics important technology </li></ul><ul><ul><li>Large-scale study of proteins </li></ul></ul><ul><ul><ul><li>Structure </li></ul></ul></ul><ul><ul><ul><li>Functions </li></ul></ul></ul><ul><li>“ Omics ” revolution: shift in strategy </li></ul><ul><ul><li>piece-by-piece -> global analysis </li></ul></ul><ul><ul><li>hypothesis-driven -> discovery-based research </li></ul></ul><ul><li>Expression proteomics </li></ul><ul><ul><li>analysis of differential protein expression </li></ul></ul><ul><li>Functional proteomics </li></ul><ul><ul><li>posttranslational modifications </li></ul></ul><ul><ul><li>protein-protein, protein-ligand interactions </li></ul></ul><ul><ul><li>sequence-structure-function relationships </li></ul></ul>Twyman RM (2004). Principles Of Proteomics. Oxford, UK: BIOS
  3. 3. Proteins <ul><li>Biochemical compounds </li></ul><ul><li>1 > polypeptides </li></ul><ul><li>Single linear polymer chain of amino acids (AA) </li></ul><ul><li>Bonded together by peptide ponds – carboxyl & AA residues </li></ul>” Proteins.&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  4. 4. Amino acid sequence ” Proteins.&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  5. 5. Protein structure Weaver, Molecular Biology, McGraw Hill: Boston, 2002
  6. 6. Proteome <ul><li>Entire set of proteins expressed by </li></ul><ul><ul><li>Genome </li></ul></ul><ul><ul><li>Cell </li></ul></ul><ul><ul><li>Tissue </li></ul></ul><ul><ul><li>Organism </li></ul></ul>> 400.000 proteins, dynamic
  7. 7. Human proteome Aebersold et al, Systems biology - ETH Zurich, https://www.e-pics.ethz.ch/
  8. 8. Why proteomics?
  9. 9. Why proteomics? <ul><li>Protein alterations cannot be fully deduced from DNA. </li></ul><ul><li>RNA expression does not always reflect protein levels: </li></ul><ul><ul><li>translational control </li></ul></ul><ul><ul><li>degradation </li></ul></ul><ul><ul><li>turnover </li></ul></ul><ul><li>Some tissues not suitable for RNA expression analysis. </li></ul><ul><li>Proteins are the physiological/pathological active key players. </li></ul><ul><li>General goal: </li></ul><ul><ul><li>better understanding of genesis and progression of diseases </li></ul></ul><ul><li>Clinical goals: </li></ul><ul><ul><li>early disease detection (biomarkers) </li></ul></ul><ul><ul><li>identification of therapeutic targets </li></ul></ul><ul><ul><li>therapy monitoring </li></ul></ul>
  10. 10. Multidisciplinary TOF, Q, IT MALDI, ESI HPLC Cells, tissue Algorithms
  11. 11. Typical stages Aebersold R, Mann M, Nature. 2003
  12. 12. Quantification strategies Domon B, Aebersold R. Science. 2006 Isotope dilution Tandem mass tags Internal standards
  13. 13. Top down or bottom up? <ul><li>Bottom-up </li></ul><ul><ul><li>Most common </li></ul></ul><ul><ul><li>Starting with proteolytic fragments </li></ul></ul><ul><ul><li>Piecing the protein back together </li></ul></ul><ul><ul><ul><li>de novo repeat detection </li></ul></ul></ul><ul><li>Top down </li></ul><ul><ul><li>Tandem MS of whole protein ions </li></ul></ul><ul><ul><ul><li>Pulling them apart </li></ul></ul></ul><ul><ul><li>Electron capture dissociation </li></ul></ul><ul><ul><li>Extensive sequence information </li></ul></ul>Fragment ions of peptides MS/MS Proteolytic digest e.g. Trypsin Protein MS/MS Fragment ions of protein Bottom-up Top down ” Protein mass spectrometry&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  14. 14. Proteomic strategies Domon B, Aebersold R. Science. 2006
  15. 15. Separation <ul><li>Specific protein biophysical parameters </li></ul><ul><ul><li>Isoelectric point </li></ul></ul><ul><ul><li>Molecular weight </li></ul></ul><ul><ul><li>Affinity </li></ul></ul><ul><li>Chromatographic methods </li></ul><ul><ul><li>HPLC </li></ul></ul><ul><ul><li>2D-HPLC </li></ul></ul><ul><ul><li>ProteinChips </li></ul></ul><ul><li>Electrophoretic methods </li></ul><ul><ul><li>SDS-PAGE </li></ul></ul><ul><ul><li>2-D E </li></ul></ul><ul><li>Reverse phase (RPLC) – Hydrophobicity </li></ul>Yates JR, et al. Annu Rev Biomed Eng. 2009
  16. 16. 2D Gel electrophoresis <ul><li>1D: isoelectric focussing (IEF) separation by IP </li></ul><ul><li>2D: dimension: SDS-PAGE separation by MW </li></ul><ul><ul><li>staining > 1000 proteins /gel </li></ul></ul><ul><li>molecular analysis by </li></ul><ul><ul><li>MS </li></ul></ul><ul><ul><li>HPLC </li></ul></ul><ul><ul><li>Westernblot </li></ul></ul><ul><li>Pitfalls </li></ul><ul><ul><li>very basic / acidic; </li></ul></ul><ul><ul><li>large / small; </li></ul></ul><ul><ul><li>hydrophobic; </li></ul></ul><ul><ul><li>low-abundance proteins </li></ul></ul>Fontana et al. Proteomics 2004
  17. 17. Robotic isolation ” Protein mass spectrometry&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  18. 18. Shotgun proteomics
  19. 20. HPLC ” HPLC&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  20. 21. HPLC <ul><li>HPLC/MS </li></ul><ul><li>Peptides from protein mixture fractionated in steps </li></ul><ul><li>Eluent </li></ul><ul><ul><li>ESI-MS </li></ul></ul><ul><ul><li>MALDI: series </li></ul></ul>” HPLC&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  21. 22. Protein & peptide fractionation <ul><li>Complex mixtures </li></ul><ul><ul><li>Proteins </li></ul></ul><ul><ul><li>Molecules </li></ul></ul><ul><li>Works only if mixture has equal amounts </li></ul><ul><ul><li>Abundant species suppress signals from less abundant ones </li></ul></ul><ul><ul><li>Difficult to interpret </li></ul></ul><ul><ul><li>Enyzmatic digestion -> many peptide products </li></ul></ul><ul><li>2-D electrophoresis </li></ul><ul><ul><li>Protein fractionation </li></ul></ul><ul><li>High performance liquid chromatography </li></ul><ul><ul><li>Peptide fractionation </li></ul></ul>” Protein mass spectrometry&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  22. 23. Proteins   Peptides  Fragments <ul><li>Proteases, e.g. trypsin, break protein into peptides </li></ul><ul><li>Tandem MS breaks peptides into fragment ions </li></ul><ul><ul><li>Measures the mass of each piece. </li></ul></ul><ul><ul><li>MS accelerates the fragmented ions; </li></ul></ul><ul><ul><li>heavier ions accelerate slower than lighter ones </li></ul></ul><ul><ul><li>MS measure mass/charge ratio of an ion </li></ul></ul><ul><ul><li>Peptides tend to fragment along the backbone. </li></ul></ul><ul><ul><li>Fragments can also loose neutral chemical groups like NH 3 and H 2 O. </li></ul></ul>H...-HN-CH-CO . . . NH-CH-CO-NH-CH-CO-…OH R i-1 R i R i+1 H + Prefix Fragment Suffix Fragment Collision Induced Dissociation
  23. 24. Ionisation <ul><li>Proteins </li></ul><ul><ul><li>Polar </li></ul></ul><ul><ul><li>Nonvolatile </li></ul></ul><ul><ul><li>Thermally unstable </li></ul></ul><ul><li>Ionisation transfers analyte into gas phase </li></ul><ul><ul><li>No degradation </li></ul></ul><ul><li>Matrix-assisted laser desorption ionization (MALDI) </li></ul><ul><ul><li>Laser nitrogen beam (soft) </li></ul></ul><ul><ul><li>Matrix protection (Sinapinic acid) </li></ul></ul><ul><li>Electrospray ionization (ESI) </li></ul><ul><ul><li>No fragmentation </li></ul></ul>MALDI
  24. 25. Mass spectrometry WE Stephens 1952 Patent
  25. 26. Mass spectrometry <ul><li>Analytical technique </li></ul><ul><ul><li>mass-charge ratio (m/z) </li></ul></ul><ul><ul><li>charged particles </li></ul></ul><ul><li>Ion source </li></ul><ul><li>Mass analyzer </li></ul><ul><li>Detector </li></ul>” Mass spectrometry&quot; Wikipedia, The Free Encyclopedia . Wikimedia Foundation, Inc.
  26. 27. Mass analysers <ul><li>Scanning and ion-beam mass spectrometers </li></ul><ul><ul><li>TOF and Q </li></ul></ul><ul><li>Trapping mass spectrometers </li></ul><ul><ul><li>IT and Orbitrap </li></ul></ul><ul><li>Whole protein mass analysis </li></ul><ul><ul><li>time-of-flight (TOF) MS, or </li></ul></ul><ul><ul><li>Fourier transform ion cyclotron resonance (FT-ICR). </li></ul></ul><ul><li>Mass analysis of proteolytic peptides more popular </li></ul><ul><ul><li>Low costs </li></ul></ul><ul><ul><li>Sample preparation is </li></ul></ul><ul><ul><li>MALDI time-of-flight instruments </li></ul></ul><ul><ul><li>Multiple stage quadrupole-time-of-flight and </li></ul></ul><ul><ul><li>quadrupole ion trap also find use in this application. </li></ul></ul>
  27. 28. Mass spectrometers
  28. 29. Base peak chromatogram Yates JR, et al. Annu Rev Biomed Eng. 2009
  29. 30. Mass chromatogram Yates JR, et al. Annu Rev Biomed Eng. 2009
  30. 31. Quantitative analysis Yates JR, et al. Annu Rev Biomed Eng. 2009 Isotopic labeling Label-free analysis
  31. 32. Quantitative proteomics Yates JR, et al. Annu Rev Biomed Eng. 2009
  32. 33. Protein identification <ul><li>Single-step? </li></ul><ul><li>Components for </li></ul><ul><ul><li>separating </li></ul></ul><ul><ul><li>identifying and quantifying the polypeptides </li></ul></ul><ul><ul><li>tools for integrating and analysing all the data </li></ul></ul><ul><li>Two main tracks. </li></ul><ul><ul><li>2DE & MS </li></ul></ul><ul><ul><li>Limited protein purification & peptide MS/MS </li></ul></ul><ul><ul><ul><li>+/- isotope tagging </li></ul></ul></ul><ul><ul><li>Efficient MS identification of gel-separated proteins </li></ul></ul><ul><ul><li>Peptide-mass mapping by MALDI-TOF </li></ul></ul><ul><ul><li>Peptide sequencing by ESI-MS/MS </li></ul></ul>Yates JR, et al. Annu Rev Biomed Eng. 2009
  33. 34. Thank you Dimitri Raptis & Alexander Koegel www.draptis.eu / proteomics.ppt
  34. 35. Proteomics Post-translational modifications using mass spectrometry www.draptis.eu/proteomics.ppt
  35. 36. Interactions & modifications Patterson & Aebersold, Nature Genetics, 2003
  36. 37. Post-translational modifications <ul><li>Any modification to the translated form of a protein </li></ul><ul><ul><li>Covalent additions that regulate protein function </li></ul></ul><ul><ul><li>determining their activity state, cellular location </li></ul></ul><ul><ul><li>dynamic interactions with other proteins </li></ul></ul><ul><li>Analysis is challenging </li></ul><ul><ul><li>Determination -> insight into biological function </li></ul></ul><ul><li>The combination of function- or structure-based purification of modified 'subproteomes', such as </li></ul><ul><ul><li>phosphorylated proteins or </li></ul></ul><ul><ul><li>modified membrane proteins </li></ul></ul><ul><ul><li>mass spectrometry </li></ul></ul>Mann & Jensen. Nature Biotechnology 2003
  37. 38. Common PTMs Mann & Jensen, Nature Biotech, 2003
  38. 39. Protein phosphorylation <ul><li>Post-translational modification of proteins </li></ul><ul><ul><li>serine, </li></ul></ul><ul><ul><li>threonine </li></ul></ul><ul><ul><li>tyrosine </li></ul></ul><ul><ul><li>residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. </li></ul></ul><ul><li>Phosphoregulation </li></ul><ul><ul><li>Phosphorylation -> most common regulation of protein function </li></ul></ul><ul><ul><li>Switches between </li></ul></ul><ul><ul><ul><li>phosphorylated -> unphosphorylated </li></ul></ul></ul><ul><ul><ul><li>active -> inactive </li></ul></ul></ul>Mann & Jensen, Nature Biotech, 2003
  39. 40. Functions of phosphorylation <ul><li>&quot;activate&quot; or &quot;energize&quot; a molecule </li></ul><ul><ul><li>to participate in a reaction with a negative free-energy change. </li></ul></ul><ul><li>Inhibit its activity </li></ul><ul><ul><li>Tyrosine kinase called &quot;src” it folds on itself </li></ul></ul><ul><ul><li>Masks its own kinase domain, and is thus shut &quot;off&quot;. </li></ul></ul>Mann & Jensen, Nature Biotech, 2003 <ul><li>Bound to other proteins which have &quot;recognition domains” </li></ul><ul><ul><li>Distinct signaling system may be activated or inhibited </li></ul></ul><ul><li>Degraded by the ATP-dependent ubiquitin/proteasome pathway </li></ul><ul><ul><li>substrates for E3 ubiquitin ligases only phosphorylated </li></ul></ul>
  40. 41. Glycosylation <ul><li>Glycosylation is the addition of saccharide to a protein or a lipid molecule </li></ul><ul><li>N-Linked Glycosylation </li></ul><ul><li>Amide nitrogen of Asparagine </li></ul><ul><li>O-Linked Glycosylation </li></ul><ul><li>Hydroxy oxygen of Serine and Threonine </li></ul>Mann & Jensen, Nature Biotech, 2003
  41. 42. Additional modifications <ul><li>Proteins subjected to: </li></ul><ul><ul><li>methylation </li></ul></ul><ul><ul><li>acetylation </li></ul></ul><ul><ul><li>glycosylation </li></ul></ul><ul><ul><li>oxidation </li></ul></ul><ul><ul><li>nitrosylation </li></ul></ul><ul><ul><li>or ALL of these modifications, </li></ul></ul><ul><ul><ul><li>time-dependent combinations </li></ul></ul></ul><ul><ul><ul><li>potential complexity one has to deal with when studying protein structure and function </li></ul></ul></ul>Jensen, Nature Reviews, 2006
  42. 43. Challenging analysis <ul><li>“ Global proteomics ” methods do not work to find PTMs </li></ul><ul><ul><li>A few modifications can be programmed into search programs – destroy MS speed and accuracy </li></ul></ul><ul><ul><li>PTMs are usually present at too low a concentration. Only the unmodified version will be seen </li></ul></ul><ul><li>Affinity enrichment techniques are required </li></ul><ul><ul><li>Affinity columns (eg., lectin cloumns, IMAC) </li></ul></ul><ul><ul><li>Antibodies </li></ul></ul><ul><ul><li>Tags (e.g., biotin) </li></ul></ul>Jensen, Nature Reviews, 2006
  43. 44. Mapping strategy - MS <ul><li>Use MS to determine PTM of isolated protein </li></ul><ul><li>Enzymatic or chemical degradation of modified protein </li></ul><ul><li>HPLC separation of peptides </li></ul><ul><li>MALDI and/or ESI used to identify PTM </li></ul><ul><li>MS/MS used to determine location of PTM(s) </li></ul>Mann & Jensen, Nature Biotech, 2003
  44. 45. Assigning PTMs - MS Jensen, Nature Reviews, 2006
  45. 46. Thank you Alexander Koegel & Dimitri Raptis www.draptis.eu / proteomics.ppt
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Proteomics

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