(Bio)chemical Proteomics. Alex Kentsis October, 2013
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1 (Bio)chemical Proteomics Alex Kentsis October,
2 A brief history of chemical proteomics 1907: Eduard Buchner, demonstration of cell-free alcohol fermentation (i.e. enzymes) 1946: James Sumner, urease crystallization (enzymes can be pure proteins) 1970: Ulrich Laemmli, protein fractionation using SDS-PAGE 1972: Christian Anfinsen, renaturation of active ribonuclease (chemical activity is related to protein conformation) 1973: Pedro Cuatrecasas with C. Anfinsen, purification of staphylococcal nuclease using aminophenyl solid support affinity chromatography (based on Lerman s purification of tyrosinase by aminophenol in 1963 and Starkenstein s purification of amylase using starch in 1910) 1973, Hugh Niall, protein sequanator for automated identification of proteins (based on Pehr Edman s amino acid cleavage in 1950) 1986, Don Hunt, protein sequencing using tandem mass spectrometry (based on Keith Jennings collision-induced dissociation) 1996, Marc Wilkins, proteomics using 2D electrophoresis and database matching
3 Mass spectrometry Thomson JJ (1913) Rays of positive electricity, Proceedings of the Royal Society, A 89, 1-20 Dempster AJ (1918) A New Method of Positive Ray Analysis, Phys. Rev. 11 (4): (discovery of 235 U) Bainbridge KT (1933) The Equivalence of Mass and Energy, Phys. Rev. 44 (2): 123. (E = mc 2 ) Lawrence EO (1945) Calutron separation of 235 U (Manhattan Project) Beckey HD (1969) Field ionization mass spectrometry Research/Development 20 (11): 26. (Soft ionization that preserves chemical structure)
4 Mass spectrometry
5 Mass spectrometry What is required? Ionization and transfer of molecules into gas phase Ion separation and detection based on mobility in vacuum in EM field
6 Ionization methods Ionization Source Acronym Event Electrospray ionization ESI evaporation of charged droplets Atmospheric pressure chemical ionization APCI corona discharge and proton transfer Matrix-assisted laser desorption/ionization MALDI photon absorption/proton transfer Desorption/ionization on silicon Fast atom/ion bombardment DIOS FAB photon absorption/proton transfer ion desorption/proton transfer Electron ionization EI electron beam/electron transfer Chemical ionization CI proton transfer
7 Electrospray ionization Surface tension vs. Electrostatic repulsion Taylor cone Electrospray wings for molecular elephants
8 Electrospray ionization Solvent evaporation Coulombic explosion Increasing Charge Density
9
10
11 Mass analyzers Mass Analyzers Magnetic Sector Time-of-Flight (TOF) Quadrupole Ion Trap Fourier Transform Ion Cyclotron Resonance MS Event magnetic field affects radius of curvature of ions time-of-flight correlated directly to ion's m/z scan radio frequency field scan radio frequency field translates ion cyclotron motion to m/z (FTMS)
12 TOF, Ion traps Ion detectors FTICR, Orbitrap R Voltage across resistor Single ion detection Ions image current detection
13 Mass spectral interpretation
14 Mass spectral resolution
15 Peptide sequencing using tandem MS Collisionally-Activated Dissociation or Collision-Induced Dissociation The molecules are accelerated into a cell filled with an inert collision gas (Ar, He, etc) Molecules undergo multiple collision, accumulating energy until they undergo chemical dissociation The peptide bond and weak side chain bonds are predominantly cleaved
16 Peptide sequencing using tandem MS y 7 y 6 y 5 y 4 y 3 y 2 y 1 O R 2 O R 4 O R 6 O R 8 H 2 N N H H N N H H N N H H N N H OH R 1 O R 3 O R 5 O R 7 O a 2 b 1 b 2 b 3 b 4 b 5 b 6 b 7 Roepstorff-Fohlmann-Biemann-Nomenclature
17 The CID Mechanism Charge-induced fragmentation
18 Peptide sequencing using tandem CID E 129 E 129 V 99 V
19 Peptide sequencing using tandem CID Monoisotopic Mass Glycine Alanine Serine Proline Valine Threonine Cysteine Isoleucine Leucine Asparagine Aspartic acid Glutamine Lysine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan
20 Relative Intensity (%) Relative Intensity (%) Protein identification using tandem MS A 100 [M+2H] T T MS C Peptid sequence tag: (684.4)ALVT(1068.6) m= Da data base search B m/z 0 [M+2H] MS/MS A I/L V T m/z T TT T EST-Sequence (ID:404211) CATTAAACTAAAAAGATGTCCCTATATGATGACCTGGGAGTGGAGACCAGTGACTCAA AAACTGAAGGCTGGTCCAAAAACTTCAAGCTCCTGCAGTCCCAGCTCCAGGTGAAGAA GGCGGCGCTCACTCAGGCCAAGAGCCAAAGGACCAAGCAAAGTACAGTGCTTGCTCCG GTCATCGACCTAAAGCGAGGCGGCTCCTCAGATGACCGGCAGATTGCAGACACACCAC CTCACGTGGCAGCTGGGCTGAAGGACCCTGTGCCCAGTGGGTTTTCTGCAGGGGAAGT TCTGATTCCCTTAGCTGA Translation into first reading frame H*TKKMSLYDDLGVETSDSKTEGWSKNFKLLQSQL QVKKAALTQAKSQRTKQSTVLAPVIDLKRGGSSDD RQIADTPPHVAAGLKDPVPSGFSAGEVLIPLA Cloning Novel protein: SPF45 SLYDDLGVETSDSKTEGWSKNFKLLQSQLQVKKAALTQAKSQRTKQSTVLAPVIDLKR GGSSDDRQIADTPPHVAAGLKDPVPSGFSAGEVLIPLADEYDPMFPNDYEKVVKRQRE ERQRQRELERQKEIEEREKRRKDRHEASGFSRRPDPDSDEDEDYERERRKRSMGGAAI APPTSLVEKDKELPRDFPYEEDSRPRSQSSKAAIPPPVYEEPDRPRSPTGPSNSFLAN MGGTVAHKIMQKYGFREGQGLGKHEQGLSTALSVEKTSKRGGKIIVGDATEKGEAQDA SKKSDSNPLTEILKCPTKVVLLRNMVGAGEVDEDLEVETKEECEKYGKVGKCVIFEIP GAPDDEAVRIFLEFERVESAIKAVVDLNGRYFGGRVVKACFYNLDKFRVLDLAEQV
21 Protein identification using tandem MS
22 Biological mass spectrometry Molecule fractionation compatible with soft ionization on mass spectrometric time-scales Reverse phase LC-MS with volatile solvents Reduction of spectral complexity and miniaturization improve sensitivity
23 LC-MS LC Column Soft ion source Mass analyzer Detector
24 High-resolution tandem MS
25 High-resolution parallel MS Dual-pressure linear ion trap Ultra-high-field Orbitrap mass analyzer Ion-routing multipole Quadrupole mass filter Active beam guide (ABG) EASY-ETD ion source
26 The One Hour Yeast Proteome
27 Relative Abundance Quantitative mass spectrometry Stable isotope detection using high-resolution mass spectrometry C %, C % N %, N % H %, H % O %, O %, O % 50 Δ1 Da Δ2 Da 0
28 Relative Abundance Quantitative mass spectrometry Lysine (light) Lysine (heavy) 100 Elution profile Time (min) Δm = +8 Da Δm/z = 4 Da Δm = +8 Da [ASVLFANEK] 2+ [ASVLFANEK] 2+
29 Ultrahigh-resolution quantitative mass spectrometry
30 Activity-based protein profiling Given chemical probe, can identify enzymatically active targets Considerations Specific vs. pleiotropic Covalent vs non-covalent Chemically and sterically permissive functional group
31 Quantitative mass spectrometry for chemical profiling
32 Quantitative mass spectrometry for chemical profiling Specificity of molecular interactions depends on concentration, e.g. all drugs are pleiotropic Biological macromolecules exist as non-covalent complexes Chemical specificity is not directly related to biological activity
33 Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90
34 Proteome-wide drug binding analysis
35 Chemical proteomics of lysine deacetylases
36 Chemical proteomic profiling using drug libraries (kinobeads)
37
38 Towards functional chemical proteomics
39 Modern biological mass spectrometry Structural characterization Elemental composition from high-accuracy mass measurements Chemical structure from gas phase fragmentation reactions Peptide sequencing using amino acid dissociation Supramolecular complex structure using soft ionization Quantitative analysis High sensitivity (zeptomole) and specificity (gold standard) ensured by direct ion detection, and selected ion and reaction monitoring (SIM and SRM) techniques Metabolites (FDA, NBS) Genotyping (Sequenom) Molecular biomarkers (Advion) Functional profiling Chemical activity using affinity chromatography coupled to MS Activity-based protein profiling (ABPP) Activity correlation proteomics Functional activity using stable isotope labeling of biological processes Stable isotope labeling in culture (SILAC)
40 Mountain of biochemical knowledge Chemistry Enzymology Structural analysis? Metabolism Cell growth and development Pleiotropy Chemical mechanism Biological function
41 Reproduction and use Attribution This presentation may contain photographs and images from other sources, which are referenced or tagged electronically Academic use You may freely use this material for academic purposes as long as you properly acknowledge it Commercial use Please contact Alex Kentsis for commercial use
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