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Proteomics Tools and Methods Outline
- 2. Outline • What is proteomics? • Why study proteins? • Discuss proteomic tools and methods
- 4. Proteomics is the analysis of the protein complement to the genome Genomics Proteomics Gene Transcript Protein
- 5. Wikipedia, http://en.wikipedia.org “..the large-scale study of proteins…while it is often viewed as the “next step”, proteomics is much more complicated than genomics. …while the genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. One organism will have radically different protein expression in different parts of its body, in different stages of its life cycle and in different environmental conditions.”
- 7. Proteomics Research •Basic research: To understand the molecular mechanisms underlying life. •Applied research: Clinical testing for proteins associated with pathological states (e.g. cancer).
- 8. Applications of Proteomics Proteomics Structural Proteomics Proteome Mining Post- translational Modifications Protein Expression Profiling Functional Proteomics Protein- protein Interactions Glycoyslation Phosphorylation Proteolysis Yeast two-hybrid Co-precipitation Phage Display Drug Discovery Target ID Differential Display Yeast Genomics Affinity Purified Protein Complexes Mouse Knockouts Medical Microbiology Signal Transduction Disease Mechanisms Organelle Composition Subproteome Isolation Protein Complexes
- 9. For example: Hemoglobin Picks up oxygen in the lungs, travels through the blood, and delivers it to the cells. O2 hemoglobin Hbβ Hbα Hbβ Hbα
- 10. ATG GTG CAC CTG ACT CCT GAG GAG … ATG GTG CAC CTG ACT CCT GTG GAG … E E M V H L T P … E V M V H L T P … Normal Hbβ Mutated Hbβ Sickle cell disease is caused by a single amino acid change.
- 11. Summary – what is proteomics? •Involves the study of proteins •Proteomics is multidisciplinary •Proteomics is being applied to both basic and clinical research
- 13. What are PROTEINS? Proteins are large, complex molecules that serve diverse functional and structural roles within cells.
- 14. Transport Hemoglobin Carries O2 Defense Antibody Fights Viruses Enzyme Protease Degrades Protein Support Keratin Forms Hair and Nails Motion Actin Contracts Muscles Regulation Insulin Controls Blood Glucose Proteins do most of the work in the cell
- 15. Proteins are comprised of amino acid building blocks R O OH C N H H Acid Base Variable CH + H2O Dipeptide Peptide Bond Amino acid 1 Amino acid 2 R1 O C C N O H H2 H R2 H C C N O O H H H R1 O C C R2 O C C H H N H2 N H OH
- 16. Asparagine Glutamate Leucine Phenylalanine Cysteine Histidine Methionine Threonine Arginine Glutamine Isoleucine Tryptophan Alanine Glycine Proline Tyrosine Aspartate Lysine Serine Valine Each amino acid has unique chemical properties. non-polar hydrophobic acidic basic polar hydrophilic
- 17. Proteins are chains of amino acids. C O OH N H H N H H Short chains of amino acids are called peptides. Proteins are polypeptide molecules that contain many peptide subunits.
- 18. G A U A U G G C C U G G 5’ 3’ Gene Messenger Ribonucleic Acid (mRNA) Amino Acid- transfer RNA Ribosome tRNA Ala tRNA Trp Met tRNA Empty tRNA Met Empty tRNA Met Ala Nucleus Cytoplasm Large Subunit Small Subunit Met Ala Trp Ribonucleotides A U G C Codon 1 A U G = Methionine C G C Codon 2 = Alanine U G G Codon 3 Tryptophan = U G Codon 4 Stop = A Translation is the synthesis of proteins in the cell.
- 20. Proteins arrive at their final structure in an ordered fashion J. E. Wampler, 1996, http://bmbiris.bmb.uga.edu/wampler/tutorial/prot0.html
- 21. Summary – why study proteins? •Biological workhorses that carry out most of the functions within the cell •Serve diverse functional and structural roles •Composed of amino acids that are covalently linked by peptide bonds •Synthesized during the translation process •Must fold correctly to perform their functions
- 22. Proteomic tools and methods
- 23. Proteomic tools to study proteins • Protein isolation • Protein separation • Protein identification
- 25. How are proteins isolated? • Mechanical Methods – grinding – break open cell – centrifugation – remove insoluble debris • Chemical Methods – detergent – breaks open cell compartments – reducing agent – breaks specific protein bonds – heat – break peptide bonds to “linearize” protein
- 26. Protein isolation procedure Find a sample Pick it Grind sample in buffer Transfer to tube Heat the sample Centrifuge to remove insoluble material “pure” protein solution Recover supernatant Keep solution for gel analysis
- 27. Protein X “pure” protein solution Isolated Protein X
- 28. Summary – protein isolation •Proteins can be isolated from a variety of samples •Proteomics includes the use of both mechanical and chemical methods to isolate proteins •Opening cell or cellular compartments •Breaking bonds and “linearizing” proteins •Removal cell debris
- 30. Why separate proteins? “PURE” Protein Solution Tube 1 Decreased Protein ID Increased Complexity Tube 2 Increased Protein ID Decreased Complexity
- 31. How to separate proteins? Separating intact proteins is to take advantage of their diversity in physical properties, especially isoelectric point and molecular weight
- 32. Methods of Protein Separation • Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE) • Isoelectric Focusing (IEF)
- 33. SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is a widely used technique to separate proteins in solution
- 34. SDS-PAGE separates only by molecular weight • Molecular weight is mass one molecule • Dalton (Da) is a small unit of mass used to express atomic and molecular masses.
- 35. PAGE is widely used in • Proteomics • Biochemistry • Forensics • Genetics • Molecular biology
- 36. Polyacrylamide gels separate proteins and small pieces of DNA • Major components of polyacrylamide gels • Acrylamide – matrix material/ NEUROTOXIN • Bis-acrylamide - cross-linking agent/ NEUROTOXINS • TEMED - catalyst • Ammonium persulfate - free radical initiator
- 37. H N H N O O Bisacrylamide (cross-linking agent) NH2 O Acrylamide (matrix material) SO4 TEMED (catalyst) Ammonium persulfate (free radical initiator) Polyacrylamide (non-toxic) Polymerization N N
- 38. Polyacrylamide (non-toxic) Polyacrylamide O NH C H2 NH O O NH C H2 NH O C ON H 2 CON H 2 C ON H 2 C ONH Bis-acrylamide cross links
- 39. Sodium dodecyl sulfate - SDS The anionic detergent SDS unfolds or denatures proteins • Uniform linear shape • Uniform charge/mass ratio
- 40. Cathode (-) Anode (+) Standard Sample1 Sample2 One-dimensional polyacrylamide gel electrophoresis (SDS-PAGE)
- 41. During SDS-PAGE proteins separate according to their molecular weight Bromophenol Blue dye front Cathode (-) Anode (+) Standard Sample1 Sample2 20 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 150 kDa
- 42. Image of Real SDS-PAG 20 kDa 250 kiloDaltons 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa Cathode Anode
- 43. Separation of Protein X Bromophenol Blue dye front Cathode (-) Anode (+) Standard Sample1 Sample2 20 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 150 kDa Protein X 11 kDa 25 kDa
- 44. Two-dimensional gel electrophoresis (2-DGE) Most widely used protein separation technique in proteomics Capable of resolving thousands of proteins from a complex sample (i.e. blood, organs, tissue…) 1st dimension - isoelectric focusing 2nd dimension - SDS-PAGE
- 45. Isoelectric focusing (IEF) is separation of proteins according to native charge. isoelectric point -pH at which net charge is zero 1st Dimension-Isoelectric Focusing
- 46. 2-DGE protein samples IEF 1st dimension SDS-PAGE 2nd dimension Neutral at pH 3 20 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 150 kDa 11 kDa pH gradient 10 3
- 47. pI mass 100 75 50 25 3 10 Arabidopsis developing leaf kDa 2-DG 4 5 6 7 8 9
- 48. 2-DGE SDS-PAGE 2nd dimension 20 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 150 kDa 11 kDa 10 3 4 5 6 7 8 9 Protein X 25 kDa pI 5
- 49. 1-DGE vs. 2-DGE 1-DGE (SDS-PAGE) • High reproduciblity • Quick/Easy • Separates solely based on size • Modest resolution, dependent on complexity of sample 2-DGE • Modest reproducibility • Slow/Demanding • Separates based on pI and size • High resolution, not dependent on complexity of sample
- 50. Summary – protein separation •Protein separation takes advantage physical properties such as isoelectric point and molecular weight •SDS-PAGE is a widely used technique to separate proteins •1-DGE is a quick and easy method to separate protein by size only •2-DGE combines isoeletric focusing (IEF) and SDS- PAGE to separate proteins by pI and size
- 52. Peptide mass fingerprinting intact protein x protein digestion mass spectrometry m/z intensity 952.0984 1895.9057 1345.6342 899.8743 2794.9761 mass Protein ID Make proteolytic peptide fragments - Digest the protein into peptides (using trypsin) Measure peptide masses - “Weigh” the peptides in a mass spectrometer Match peptide masses to protein or nucleotide sequence database - Compare the data to known proteins and look for a match
- 53. Protein digestion We use the enzyme TRYPSIN to digest (cut) proteins into peptides – trypsin cuts after Lysine (K) and Arginine (R) ????????K?????R???????? ????????K?????R???????? ????????K?????R???????? Protein X ????????K?????R???????? ????????K?????R???????? ????????K?????R????????
- 54. How does mass spectrometry identify unknown proteins?
- 55. Basics of mass spectrometry • determination of mass to charge ratio (m/z) • Mass spectrometer = very accurate weighing scales – third or fourth decimal place
- 56. ????????K ?????R ???????? We then “weigh” these peptides with a Mass Spectrometer Mass Spectrometer
- 57. ????????K ?????R ???????? We then “weigh” these peptides with a Mass Spectrometer 692.31 Da 1106.55 Da 1002.37Da
- 58. Mass of peptides should be compared to theoretical masses of known peptides ?????R = 692.31 Da ????????K = 1106.55 Da ???????? = 1002.37Da
- 59. Computation of theoretical masses of known peptides known Computer Peptides • WEGETMILK 1106.55 • ADEMTYEK 1105.23 • PLMEHGAK 1089.50 • LMEHHH 782.25 • ASTEER 692.31 • DMGEYIILES 1056.92 • EGEDMPAFY 1002.35 • CYHGMEI 984.36 • EFPKLYSEK 900.56 • YSEPYSSIIR 1102.34 • IESPLMIA 864.35 • AEFLYSR 600.21 • DLMILIYR 864.97 • METHIPEEK 795.36 • KISSMER 513.21 • PEPTIDEK 456.23 • MANYCQWS 792.15 • TYSMEDGHK 678.46 • YMEPSATFGHR 995.46 • GHLMEDFSAC 896.35 • HHFAASTR 564.88 • ALPMESS 469.12 Proteome = all protein sequences Digest Proteome with simulated Trypsin
- 60. Mass of peptides compared to theoretical masses of all peptides known, using a computer program. ?????R = 692.31 Da ????????K = 1106.55 Da ???????? = 1002.37Da Computer Peptides • WEGETMILK 1106.55 • ADEMTYEK 1105.23 • PLMEHGAK 1089.50 • LMEHHH 782.25 • ASTEER 692.31 • DMGEYIILES 1056.92 • EGEDMPAFY 1002.35 • CYHGMEI 984.36 • EFPKLYSEK 900.56 • YSEPYSSIIR 1102.34 • IESPLMIA 864.35 • AEFLYSR 600.21 • DLMILIYR 864.97 • METHIPEEK 795.36 • KISSMER 513.21 • PEPTIDEK 456.23 • MANYCQWS 792.15 • TYSMEDGHK 678.46 • YMEPSATFGHR 995.46 • GHLMEDFSAC 896.35 • HHFAASTR 564.88 • ALPMESS 469.12
- 61. Mass of peptides matched to theoretical masses known peptides, using a computer program. ?????R = 692.31 Da ????????K = 1106.55 Da ???????? = 1002.37Da Computer Peptides • WEGETMILK 1106.55 • ADEMTYEK 1105.23 • PLMEHGAK 1089.50 • LMEHHH 782.25 • ASTEER 692.31 • DMGEYIILES 1056.92 • EGEDMPAFY 1002.35 • CYHGMEI 984.36 • EFPKLYSEK 900.56 • YSEPYSSIIR 1102.34 • IESPLMIA 864.35 • AEFLYSR 600.21 • DLMILIYR 864.97 • METHIPEEK 795.36 • KISSMER 513.21 • PEPTIDEK 456.23 • MANYCQWS 1002.37 • TYSMEDGHK 678.46 • YMEPSATFGHR 995.46 • GHLMEDFSAC 896.35 • HHFAASTR 564.88 • ALPMESS 469.12
- 62. The unknown peptides have been identified ?????R = 692.31 Da ????????K = 1106.55 Da ???????? = 1002.37Da WEGETMILK ASTEER MANYCQWS
- 63. Protein X has been identified ????????K?????R???????? ????????K?????R???????? ????????K?????R???????? WEGETMILK AFTEER MANYCQWS
- 64. Summary – tools to study proteins? •Proteins are digested into peptides •Peptides are analyzed with a mass spectrometer •Match observed peptide masses to theoretical masses of all peptides in database •Assemble those peptide matches into a protein identification
- 65. Concluding points about Proteomics -Proteomics is the analysis of all proteins -Interdisciplinary research -Essential to both basic and clinical research -Protein are the workhorses of the cell - Discovery research – drugs and diseases -Proteomics tools allow identification of proteins
- 66. Questions