(Bio)chemical Proteomics Alex Kentsis October, 2013 http://alexkentsis.net
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
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): 316 325 (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)
Mass spectrometry
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
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
Electrospray ionization + - + - + + + + + + + + + + + + + + Surface tension vs. Electrostatic repulsion Taylor cone Electrospray wings for molecular elephants http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2002/fenn-lecture.html
Electrospray ionization + + + + + + + + + + + + + + + + + + Solvent evaporation Coulombic explosion + + + + + + + + + + Increasing Charge Density http://pubs.acs.org/doi/abs/10.1021/ac00070a001
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)
TOF, Ion traps Ion detectors FTICR, Orbitrap R Voltage across resistor Single ion detection Ions image current detection
Mass spectral interpretation
Mass spectral resolution
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
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
The CID Mechanism Charge-induced fragmentation
Peptide sequencing using tandem CID 986.593 1244.702 899.013 1115.644 1343.766 E 129 E 129 V 99 V 99 1442.865
Peptide sequencing using tandem CID Monoisotopic Mass Glycine 57.02147 Alanine 71.03712 Serine 87.03203 Proline 97.05277 Valine 99.06842 Threonine 101.04768 Cysteine 103.00919 Isoleucine 113.08407 Leucine 113.08407 Asparagine 114.04293 Aspartic acid 115.02695 Glutamine 128.05858 Lysine 128.09497 Glutamic acid 129.0426 Methionine 131.04049 Histidine 137.05891 Phenylalanine 147.06842 Arginine 156.10112 Tyrosine 163.06333 Tryptophan 186.07932
Relative Intensity (%) Relative Intensity (%) Protein identification using tandem MS A 100 [M+2H] 2+ 642.5 T T MS C Peptid sequence tag: (684.4)ALVT(1068.6) m=1283.0 Da data base search B 100 0 400 500 600 700 800 m/z 0 [M+2H] 2+ 642.5 684.4 MS/MS A I/L V T 1068.6 200 400 600 800 1000 1200 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
Protein identification using tandem MS
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
LC-MS LC Column Soft ion source Mass analyzer Detector
High-resolution tandem MS
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
The One Hour Yeast Proteome http://www.ncbi.nlm.nih.gov/pubmed/24143002
Relative Abundance Quantitative mass spectrometry Stable isotope detection using high-resolution mass spectrometry C 12 98.9 %, C 13 1.10 % N 14 99.63 %, N 15 0.37 % H 1 99.985 %, H 2 0.015 % O 16 99.76 %, O 17 0.04 %, O 18 0.20 % 50 Δ1 Da Δ2 Da 0
Relative Abundance Quantitative mass spectrometry Lysine (light) Lysine (heavy) 100 Elution profile 0 18.4 18.8 19.2 19.6 20.0 20.4 Time (min) 100 489.77 490.27 493.77 Δm = +8 Da Δm/z = 4 Da Δm = +8 Da 494.27 490.77 493.27 494.77 491.77 0 489.5 490.5 491.5 492.5 493.5 494.5 495.5 [ASVLFANEK] 2+ [ASVLFANEK] 2+
Ultrahigh-resolution quantitative mass spectrometry http://www.nature.com/nmeth/journal/v10/n4/full/nmeth.2378.html
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 http://dx.doi.org/10.1016/j.cbpa.2003.11.004
Quantitative mass spectrometry for chemical profiling http://www.pnas.org/content/106/12/4617.abstract
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
Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90 http://www.nature.com/nchembio/journal/v7/n11/full/nchembio.670.html
Proteome-wide drug binding analysis http://www.nature.com/nbt/journal/v29/n3/full/nbt.1759.html
Chemical proteomics of lysine deacetylases
Chemical proteomic profiling using drug libraries (kinobeads) http://www.nature.com/nbt/journal/v25/n9/full/nbt1328.html
Towards functional chemical proteomics http://www.ncbi.nlm.nih.gov/pubmed/18662549
Modern biological mass spectrometry Structural characterization Elemental composition from high-accuracy mass measurements Chemical structure from gas phase fragmentation reactions http://chemdata.nist.gov 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)
Mountain of biochemical knowledge Chemistry Enzymology Structural analysis? Metabolism Cell growth and development Pleiotropy Chemical mechanism Biological function
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