PAF_PROTEOMICS_INTRO.ppt

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Slide 1: PAF introduction Slide 2: The PAF (also called UNIL Proteomics Platform) is a research and service core facility devoted to the high-performance analysis of proteins •Service : •Identification of gelseparated proteins by MS •Project discussion •2D-PAGE and/or other multi-dimensional separation techniques Research : •Development of proteomics methods •Applications to biological problems •Teaching : •Techniques, possibilities & limitations of proteomics approaches Slide 3: PAF technology and instrumentation 260404RPChistonesFRAC001:1_UV1_280nm 260404RPChistonesFRAC001:1_Inject mAU 30.0 260404RPChistonesFRAC001:1_Conc 260404RPChistonesFRAC001:1_Fractions 20.0 10.0 0.0 -10.0 -20.0 -30.0 A1 0.0 A2 A3 A4 A5 A6 A7 A8 A9A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 D12 D11 D10 D9 5.0 10.0 15.0 20.0 25.0 ml 1D-electrophoresis 2D-electrophoresis Liquid Chromatography Nano-HPLC- Quadrupole-time-of-flight Mass spectrometer Nano-HPLC-triple quadrupole ion trap Mass spectrometer Slide 4: New : access to ABI 4700 TOF/TOF™ (courtesy Dept. Biochemistry) MALDI - Tandem Time of Flight Mass Spectrometer for High Throughput Protein identification Slide 5: Challenges of in vivo proteomics • Complexity : – 35’000 genes (?) in H. sapiens, – 15’000 expressed in a single cell ? – but >50’000 chemically different protein species ? • Dynamic range : – 105 x or 106 x between low and high-abundance proteins • Plasticity : – continuous variation in protein expression pattern (every s), PTM’s, degradation,… Slide 6: Overview : classes of proteomics experiments Protein expression analysis FOCUS : Complex samples Whole proteomes 200 and more proteins Interaction / Functional Proteomics FOCUS : Subcellular fraction Organelle Protein Complex 20-200 proteins FOCUS : Single protein PTM analysis 1-20 proteins Analytical Detail Sample complexity Slide 7: Proteomics • 2D-PAGE • Mass Spectrometry for proteomics • Proteomics workflows and applications Slide 8: 2D-PAGE Slide 9: IEF: the principle How to create a pH gradient ? Slide 10: Improvements in 2D-PAGE + pH 3.0 10.0 - - IPG (Immobilised Ph Gradient) strips for the first dimension pH-forming chemical groups are grafted onto the polyacrylamide matrix, creating a mechanically stable pH gradient ++ ++ ++ ++ mechanical stability reproducibility loadable amounts „zoom“ pI ranges Slide 11: After IEF : equilibration and 2nd dimension + - pI Equilibration step 1 : •SDS •Buffer pH 6.8 •DTT (reduce –S-S-) Equilibration step 2 : •SDS •Buffer pH 6.8 •Iodoacetamide (alkylate –S-S-) 3.0 5.5 6.5 8.5 10 150 100 75 50 37 20 10 Slide 12: One protein  many spots Post-translational modifications can result in changes in pI and/or MW Spot “trains” for extensively modified proteins Caused by Glycosylation Phosphorylation Acetylation (K) … Database of 2D images with clickable spots : www.expasy.org/ch2d/ Slide 13: 2D-PAGE and image analysis are used for studying changes in composition of the proteome Slide 14: Control Stimulus applied Slide 15: Software-based image analysis •Spot detection •Spot quantification •Gel-to-gel •Matching •Presence/absence of spot •Up/down regulation •Statistic analysis Slide 16: Mass Spectrometry in proteomics Slide 17: Mass spectrometry : essential functions ION SOURCE ION GENERATION MASS ANALYZER ION SEPARATION DETECTOR ION DETECTION SAMPLE ESI : Electrospray Ionisation MALDI : Matrix Assisted Laser Disorption/Ionization Quadrupoles Ion traps Time-of-flight with reflectron TOF/TOF FT-ICR (Fourier transform – Ion Cyclotron Resonance) Faraday cup Scintillation counter Electromultiplier High-energy dynodes with electronmultiplier Array (detector) FT-MS Slide 18: Masses and mass measurements •All mass spectrometers function measure molecules in their ionized state •All values determined by MS are relative to the m/z assumed by the molecule after the ionization process The relationship between the molecular mass (m) and the m/z value can be calculated as follows: m/z = (m + (mA * z )) / z mA is the mass of the adduct responsible for ionization (typically H+ for positive MS mode). Slide 19: MALDI IONISATION MALDI (Matrix Assisted Laser Desoprtion Ionisation MALDI TOF (Time Of Flight) Slide 20: MALDI TOF • Great for Peptide Mass Fingerprinting – Fast – Easy to measure – Sensitive – Salt-tolerant (to some extent) – Also good for larger MW (small proteins) – Sample on a stable support (no time constraints) – 1+ ions  simpler data analysis •High accuracy needs careful calibration •Difficult (but possible) to do MS/MS by MALDI •Signal suppression in complex mixtures •Crystallisation conditions influence results disadvantages Slide 21: MALDI-TOF of a tryptic digest of BSA +TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ... a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded 927.59 190 180 170 160 150 140 130 In t e n s ity , c o u n ts 120 110 100 90 80 70 60 50 40 30 20 789.53 10 0 800 871.07 857.14 900 1024.56 978.60 1000 1050.55 1073.03 Max. 1305.0 counts. YLYEIAR LGEYGFQNALIVR LVNELTEFAK HPEYAVSVLLR HLVDEPQNLIK 1440.00 LSQKFPK 847.59 ? DAFLGSFLYEYSR 1479.98 1567.94 1640.16 ? 869.07 ? 1022.56 FKDLGEEHFK KVPQVSTPTLVEVSR 1163.77 1305.87 1296.86 1283.91 1292.95 1300 m/z, amu 1481.98 ? 1249.77 1142.86 1108.71 1100 1200 1417.93 1443.01 1386.76 1501.84 1400 1500 ? 1595.95 1616.92 1790.10 1600 1700 1824.09 1800 ? Slide 22: ELECTROSPRAY IONISATION • molecules compete for ionisation e.g. Na+ >> Peptide P. Kebarle, M. Peschke / Analytica Chimica Acta 406 (2000) 11–35 Slide 23: ELECTROSPRAY IONISATION • Great for MS/MS – Can be directly coupled to reversed phase LC (separation !) – Sensitive – Excellent for MS/MS due to 2+/3+ ions disadvantages •Sample introduction more complex •Data analysis more difficult (2+/3+ ions) •one-shot sample analysis (time constraints) •Very low tolerance to contaminants Slide 24: 1+ versus 2+ ions 84 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 1160 1162 1163.76 200 180 582.28 ∆m=1.0 Da 160 140 ∆m=0.5 Da 582.77 1164.76 120 100 80 60 1165.74 Intensity, counts Intensity, counts 1.0 Da 1.0 Da 0.5 Da 583.27 40 1166.78 1162.70 20 0 580.0 0.5 Da 1164 1166 1168 m/z, amu 1170 1172 1174 581.0 582.0 583.0 584.0 m/z, amu 585.0 586.0 1+ 2+ Slide 25: Modes of measurement : MS & MS/MS •Ion production (ionisation) •Ion separation •Ion detection •Ion production (ionisation) •Ion separation – isolation of “parent” ion •Ion fragmentation (CID) •Ion separation – separate fragment ions •Ion detection - measure fragment ions MS Tandem MS MS/MS Slide 26: Tandem MS (MS/MS) facts • MS/MS results in the acquisition of pseudo-sequence information for multiple (as many as possible) tryptic peptides in addition to intact peptide mass information • MS/MS needs an instrument able to perform ion isolation and fragmentation, in addition to regular MS •• MS/MS results in the acquisition of multiple orthogonal data files (1 CID= collision induced dissociation spectrum / peptide) •Low energy (<100 eV) vs high energy collisions (>> 100 eV) •Precursor ion = parent ion : the one being fragmented •Daughter ions = fragment ions produced by CID •Tandem mass spectrometry = MS/MS Slide 27: MS/MS Glossary and facts •CID= collision induced dissociation •Low energy (<100 eV) vs high energy collisions (>> 100 eV) •Precursor ion = parent ion : the one being fragmented •Daughter ions = fragment ions produced by CID •Tandem mass spectrometry = MS/MS •- here : the combination of ion selection / CID / fragment analysis •ESI of tryptic peptides typically generates doubly charged ions due to the presence of Lys or Arg at the C terminal end of the peptides •y and b-ion series fragments are usually observed in MS/MS fragmentation spectra. Slide 28: Covalent bonds being broken  ion series Slide 29: Covalent bonds being broken  ion series Slide 30: ESI & Quadrupole-based instruments Triple Quadrupole Triple QuadrupoleIon trap Quadrupole-Quadrupole TOF Ion Trap (3D trap) All these instruments can perform MS/MS fragmentation experiments Slide 31: • Identifying proteins by mass spectrometry Slide 32: MS-based protein identification : general concept experimental In silico Protein sample Specific protease e.g. trypsin Protein sequence(s) software Protein fragments (5-30 AA peptides) MS Protein fragment sequences (same protease specificity) software Exact masses of peptides Fragmentation (MS/MS) spectrum of each peptide software Calculated exact masses of peptides Calculated fragmentation spectrum of each peptide Best Match(es) Slide 33: • Protein identification by Peptide Mass Fingerprinting (PMF) Slide 34: Information contained in MS spectrum Extracted peak list m/z 847.5896 869.0722 922.5712 923.5815 927.5904 1022.5551 1050.5533 1163.7695 1164.7531 1193.7393 1249.7705 1250.8103 1296.8556 1297.8499 1305.8668 1416.8929 1440.0008 1479.9773 1482.9583 1567.9417 1640.1635 1824.06 … +TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ... a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded 927.59 190 180 170 160 150 140 130 I n t e n s it y , c o u n t s 120 110 100 90 80 70 60 50 40 30 20 789.53 10 0 800 871.07 857.14 900 1024.56 978.60 1000 1249.77 1050.55 1073.03 1142.86 1108.71 1100 1200 869.07 1022.56 1163.77 1305.87 1296.86 1283.91 1417.93 1386.76 1443.01 1595.95 1824.09 1292.95 1501.84 1616.92 1790.10 1300 m/z, amu 1400 1500 1600 1700 1800 1481.98 847.59 1440.00 1479.98 1567.94 1640.16 Max. 1305.0 counts. Slide 35: Search form… Slide 36: Results page… Slide 37: • Protein identification by MS/MS Slide 38: Experimental set-up : nanoLC-MS/MS HPLC pumps ~95% further analysis (waste) ~5 % T-splitter sample Mass spectrometer C18 Column L = 10 cm ID = 50-100 µm database correlation Slide 39: An on-line LC-ESI-MS experiment with automatic data acquisition 568.58 3.6e4 3.2e4 2.8e4 445.07 496.57 354.11 563.23 MeCN gradient 461.71 Mr peptide : 921.4 Intensity, cps Intensity, counts 2.4e4 2.0e4 1.6e4 1.2e4 8000.0 4000.0 0.0 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Time, min +TOF Product (461.7) 86.09 20 K 15 147.01 I D y2 F L y5 635.31 I S S y1 10 175.06 260.17 270.11 y4 522.23 y3 5 375.19 359.01 y6 748.39 y7 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 y8 m/z, amu Slide 40: Automated LC- MS/MS run Chromatogram : Total ion current vs. time 1.00e5 421.68 653.32 722.28 5.00e4 464.17 501.73 582.25 507.75 595.29 740.41 700.34 0.00 25 30 35 Full scan : +TOF MS: 400 1 40 45 50 Time, min 55 60 65 70 75 80 1553 (3+) 518.82 4 200 507.1541 0 400 450 500 531.47 536.80 2 1553 (2+) 777.8176 3 662.76 MS/MS peptide 4 : +TOF Product (662.8): 14.0 10.0 550 600 650 700 750 800 850 m/z, amu 900 235.08 166.06 147.10 120.06 110.04 138.06 100 207.09 262.10 409.18 614.2136 663.3252 5.0 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z, amu 1300 Slide 41: Matching of MS / MS data b2 19.7 19.0 18.0 17.0 16.0 15.0 14.0 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 I 4.0 86.09 I 3.0 2.0 1.0 0.0 y2 b1 138.05 y1 332.21 Intensity, counts a1 I 110.06 a2 223.13 251.12 +TOF Product (653.3) Residue Immonium a b y ----------------------------------------------------------------------------------H, His 110.07 110.07 138.06 1305.71 L, Leu 86.09 223.15 251.15 1168.65 V, Val 72.08 322.22 350.21 1055.57 D, Asp 88.03 437.25 465.24 956.50 E, Glu 102.05 566.29 594.28 841.47 P, Pro 70.06 663.34 691.34 712.43 Q, Gln 101.07 791.40 819.39 615.38 N, Asn 87.05 905.44 933.44 487.32 L, Leu 86.09 1018.53 1046.52 373.28 I, Ile 86.09 1131.6157 1159.61 260.19 K, Lys 101.10 1259.7106 1287.70 147.11 MH22+ precursor 653.36 b5 594.25 Black : predicted Red : predicted and detected b3 y6 y9 350.24 712.46 a5 1055.49 y10 y3 b4 y7 y8 566.32 373.30 465.16 166.05 841.53 956.41 1168.56 b8 206.16 200 300 400 500 600 700 800 m/z, amu 900 1000 1100 1200 1300 100 Slide 42: Mascot search output Mascot Search Results Significant hits: ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). ALBU_CANFA (P49822) Serum albumin precursor (Allergen Can f 3). VWF_PIG (Q28833) Von Willebrand factor precursor (vWF) (Fragment). CIQ3_BOVIN (P58126) Voltage-gated potassium channel protein KQT-like 3 RYR2_RABIT (P30957) Ryanodine receptor 2 (Cardiac muscle-type ryanodin K2CA_BOVIN (P04263) Keratin, type II cytoskeletal 68 kDa, component IA ALFB_RABIT (P79226) Fructose-bisphosphate aldolase B (EC 4.1.2.13) (Li ALBU_BOVIN Mass: 71244 Total score: 711 Peptides matched: 12 (P02769) Serum albumin precursor (Allergen Bos d 6). Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide 15 19 31 33 47 58 60 61 64 74 97 34 98 461.80 921.58 921.48 0.10 501.80 1001.58 1001.58 0.01 569.80 1137.58 1137.49 0.09 582.30 1162.58 1162.62 -0.04 653.40 1304.78 1304.71 0.08 722.40 1442.78 1442.63 0.15 739.80 1477.58 1477.52 0.07 740.40 1478.78 1478.79 -0.00 751.90 1501.78 1501.61 0.18 547.30 1638.88 1638.93 -0.05 627.70 1880.08 1879.91 0.16 1 RPCFSALTPDETYVPK 636.70 1907.08 1906.91 0.16 0 0 0 0 0 0 0 0 0 1 0 0 55 31 72 76 90 87 43 61 37 85 1 1 1 1 1 1 1 1 1 1 AEFVEVTK LVVSTQTALA CCTESLVNR LVNELTEFAK HLVDEPQNLIK YICDNQDTISSK ETYGDMADCCEK LGEYGFQNALIVR EYEATLEECCAK KVPQVSTPTLVEVSR 42 1 LFTFHADICTLPDTEK Slide 43: Orthogonal datasets and confidence levels PMF : one MS spectrum  one dataset (peak list) MS/MS : n MS/MS spectra  n orthogonal datasets Database : 100’000 sequences 500 spectra Probability of one (any) spectrum “accidentally” matching a sequence (wrong match) : 1/100’000 x 500 = 5.10-3 (0.005) Probability of 2 spectra “accidentally” matching the same sequence (wrong match) : 5.10-3 x 5.10-3 = 2.5 x 10-5 Much higher confidence of identification with at least two peptides matching the same protein sequence Every peptide is unambiguously assigned to its “parent “ sequence, therefore many proteins can be identified in one sample during one run Slide 44: Biological question Summary : Typical Analytical Workflow Chromatographic Separation (reversed-phase) Protease digestion Peptide extraction Nano-HPLC time Tandem mass spectra of 502000 peptides m/z MS/MS Output : •Protein identification in simple/complex mixtures •Extensive sequence coverage and peptide mapping •Analysis of modified peptides possible Database searching Software (MASCOT) Protein sequence database Database matches DHX9_HUMAN ATP-dependent RNA helicase A NFM_HUMAN Neurofilament triplet M protein Q9BQG0 Hypothetical protein MYO6_HUMAN Myosin VI. TP2A_PIG DNA topoisomerase II, alpha isozyme Q7Z5Y2 Rho-interacting protein 3. FLIH_HUMAN Flightless-I protein homolog. TP2B_MOUSE DNA topoisomerase II, beta isozyme S3B1_HUMAN Splicing factor 3B subunit Q8VCW5 Similar to alpha internexin neuronal Q8CHF9 MKIAA0376 protein (Fragment). Q7Z5Y2 Mass: 118789 Total score: 178 Peptides matched: 6 Rho-interacting protein 3. Mr(calc) Score Peptide 930.48 42 EGLTVQER 1032.54 11 NWIQTIMK 1206.63 29 FSLCILTPEK 1369.75 24 LSTHELTSLLEK 1406.77 55 FFILYEHGLLR 1775.88 16 QVPIAPVHLSSEDGGDR Slide 45: Caveat : Protein identification IS NOT protein characterisation Two peptides are enough to identify a protein But We are still identifying two peptides, not the entire protein Highly similar sequences cannot be distinguished For finding PTMs extensive Sequence coverage is essential !! Slide 46: 2 Technology, workflows and applications : what is available Slide 47: New and old tools Genome sequence databases Protein separation techniques - Liquid chromatography - Electrophoresis -… Protein identification techniques - Mass Spectrometry - Antibody-based techniques Protein quantification techniques - Antibody based techniques - dye-binding techniques - Mass Spectrometry Protein sequence databases Biological knowledgebases : - functions - pathways - seq. motifs - 3D structures Slide 48: WORKFLOWS 1 : „classical“ 2D-PAGE + MALDI TOF Slide 49: Workflow 1 : adaptation of bacteria to growth conditions Normal medium 1 3A 3B 87 9 12 2 4 56 Low Glucose 1 3A 3B 87 9 12 2 4 6 10 11 10 11 15 8 14 E.Coli adapts to a very low glucose medium by up- and downregulating a set of 15 proteins Wick LM, et al, Environ Microbiol 3: 588-599, 2001 Slide 50: Workflow 1 : adaptation of bacteria to growth conditions M5 M3 M2 M1 Tryptic digestion M8 M4 M6 M7 Peptide Mass Fingerprinting Search with mass list M1......M8 Nr 1 2 3 4 5 6 7 8 9 mw* 52.2 60.3 47.5 48.45 55.2 ? 41.3 40.7 33.36 pI* 5.07 6.21 5.06 6.71 6.02 ? 7.03 5.22 5..25 Acc. N. P25553 P23847 P05313 P10904 P00822 P76108 P02917 P02928 P02927 Name aldA dppA aceA ugpB atpA ydcS livJ malE mglB Function central metabolism peptide transport Central metabolism transport proton transport/energy transport amino acid transport sugar transport sugar transport Metabolic enzymes and transport proteins affected Utilisation of alternative sources of energy Slide 51: WORKFLOW 1 : quantitation and kinetics 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 0.12 0.10 0.08 0.06 0.04 0.02 0 14 12 10 8 6 4 2 0 3.0 2.5 2.0 1.5 1.0 0.5 0 AldA (1) 12345 UgpB (4) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 18 16 14 12 10 8 6 4 2 0 0.25 0.20 0.15 0.10 0.05 0 DppA (2) 12345 AtpA (5) 6 5 4 3 2 1 0 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 AceA (3A) 12345 YdcS (6) 1.2 1.0 0.8 0.6 0.4 0.2 0 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 AceA (3B) 12345 LivJ (7) 12345 MalE (8) 12345 MglB (9) 12345 ArgT (12) 12345 MalI (13) 12345 3.0 GatY (10) 2.5 2.0 1.5 1.0 0.5 0 12345 12345 RbsB (11) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 12345 Time points : 1) Start – batch culture 2) Adapted bacteria put back into highglucose batch culture 3) 40 hr adaptation culture 4) 156 hr adaptation culture 5) 500 hr adaptation culture Relative protein quantities as a function of time during the adaptation process 12345 12345 Slide 52: Why is SDS-PAGE such a good preparation method? • • • • • • Ideal interface to biology Analytical and micropreparative Robust Solid phase chemistry of proteins Easy, low-tech Removal of contaminants : – At the loading point – After migration during fix / staining steps Disadvantages • protein digestion in gel: non quantitative • peptide sequence recovery: usually incomplete • whole protein recovery: poor Slide 53: In-gel digestion: solid phase chemistry of proteins Slide 54: WORKFLOWS 2 : General shotgun protein identification techniques example : Affinity pull-down + 1D-PAGE + LC-MS/MS Slide 55: Shotgun sequencing from complex mixtures Denaturation, Proteolytic digestion Multiprotein complex Complex peptide mixture (1000-20000 species) List of identified proteins 1. 2. 3. 4. 5. 6. P45218 P21543 Q12588 P32651 Q01245 …. Rp-LC-MSMS run Db search Slide 56: Alternative : MuDPIT (Multi Dimensional Protein Identification Technology) Post-digestion separation of peptides by two-dimensional liquid chromatography instead of separation of proteins Strong Cation Exchange (SCX) separation Multiprotein mixture (complex) Denaturation, Proteolytic digestion Complex peptide mixture (1000-20000 species) List of identified proteins 1. 2. 3. 4. 5. 6. P45218 P21543 Q12588 P32651 Q01245 …. Db search Rp-LC-MSMS runs Slide 57: A VERY complex mixture – direct analysis (no separation) TIC: from 151002_ACO_B4strep.wiff 5.3e5 5.0e5 526.26 4.5e5 303.13 4.0e5 3.5e5 In te n s ity , c p s 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 0.0 402.54 541.11 574.66 472.25 536.34 1064.42 494.25 330.18 86.10 406.23 852.44 199.19 203.11 186.12 86.10 629.30 653.36 133.06 Max. 5.3e5 cps. 449.15 665.38 527.26 615.41 563.30 599.32 409.18 5 10 15 20 25 30 35 40 45 50 55 60 65 Time, min 70 75 80 85 90 95 100 105 110 115 Slide 58: A VERY complex mixture still gives results, but... Mascot Search Results User : MQ Email : Search title :151002_ACO_B4strep.wiff : Angelos frac B4 IP strept MS data file : C:\DOCUME~1\paf\LOCALS~1\Temp\mas5D.tmp Database : Sprot 4028 (114033 sequences; 41888693 residues) Taxonomy : Mammalia (mammals) (23838 sequences) Timestamp : 17 Oct 2002 at 08:09:30 GMT Significant hits: ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). DNM1_HUMAN (P26358) DNA (cytosine-5)-methyltransferase 1 (EC 2.1.1.37) AC15_HUMAN (P35251) Activator 1 140 kDa subunit (Replication factor C IF16_HUMAN (Q16666) Gamma-interferon-inducible protein Ifi-16 (Interfe K1CJ_HUMAN (P13645) Keratin, type I cytoskeletal 10 (Cytokeratin 10) ( K22E_HUMAN (P35908) Keratin, type II cytoskeletal 2 epidermal (Cytoker ACF7_HUMAN (Q9UPN3) Actin cross-linking family protein 7 (Macrophin) ( AC14_HUMAN (P35250) Activator 1 40 kDa subunit (Replication factor C 4 ALBU_FELCA (P49064) Serum albumin precursor (Allergen Fel d 2). AC15_MOUSE (P35601) Activator 1 140 kDa subunit (Replication factor C DYHC_MOUSE (Q9JHU4) Dynein heavy chain, cytosolic (DYHC) (Cytoplasmic AC12_HUMAN (P35249) Activator 1 37 kDa subunit (Replication factor C 3 EF11_CRIGR (P20001) Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elonga RYR3_HUMAN (Q15413) Ryanodine receptor 3 (Brain-type ryanodine recepto K2C1_HUMAN (P04264) Keratin, type II cytoskeletal 1 (Cytokeratin 1) (K PLE1_RAT (P30427) Plectin 1 (PLTN) (PCN). AHNK_HUMAN (Q09666) Neuroblast differentiation associated protein AHNA TRYP_PIG (P00761) Trypsin precursor (EC 3.4.21.4). ACF7_MOUSE (Q9QXZ0) Actin cross-linking family protein 7 (Microtubule CENF_HUMAN (P49454) CENP-F kinetochore protein (Centromere protein F) ALBU_HUMAN (P02768) Serum albumin precursor. PLE1_HUMAN (Q15149) Plectin 1 (PLTN) (PCN) (Hemidesmosomal protein 1) TRI4_HUMAN (Q15650) Activating signal cointegrator 1 (ASC-1) (Thyroid NF1_HUMAN (P21359) Neurofibromin (Neurofibromatosis-related protein N NEBU_HUMAN (P20929) Nebulin ... Slide 59: ........ how deep are we going ? 2.7e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 120.08 157.15 545.75 545.75 681.35 498.55 569.27 489.53 445.07 445.10 18 20 22 24 26 28 30 32 34 36 38 40 42 581.28 652.36 216.11 670.84 131.09 343.18 738.39 555.22 153.08 561.29 548.74 Intensity, cps Intensity, counts 499.72 499.70 469.25 469.24 651.85 445.09 648.38 46 48 44 Time, min 180 160 140 120 100 80 60 40 20 10 0 analyzed missed 450 500  some protein prefractionation 550 600 650 is necessary ! 700 750 800 850 900 m/z, amu Slide 60: TNF family of ligands and TNF-receptor family Bodmer JL et al, TIBS. 2002 27(1):19-26 Slide 61: Analysis of apoptotic signalling complexes The Fas (CD95) signalling complex (DISC) (-) FasL (+) FasL ? Fc-FasL Casp.10 Casp.8 Flip ? Fas FADD Model Western blot Real life Slide 62: Analysis of apoptotic signalling complexes : negative control (-) BAFF (+) FasL (-) BAFF (+) FasL 1 cm * run 1 cm * no fix, no stain ! * cut, digest Slide 63: LC-MS/MS HL search results FasL ANALYSIS TIC: from 180702BaffHL.wiff 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 I n t e n s it y , c p s I n t e n s it y , c p s 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 5 10 15 20 25 30 BAFF HL search results Database : MSDB 200402 (851746 sequences; 265326103 residues) : MSDB 200402 (851746 sequences; TaxonomyDatabase : Mammalia (mammals) (166849 sequences) 265326103 residues) Taxonomy : Mammalia (mammals) (166849 sequences) BAFF complex HL sample Significant hits: Significant hits: A37241 52K autoantigen Ro/SS-A - human Max. 3.8e5 cps. LCMS runs Fas/Baff FasL complex HL sample TIC: from FAS_180702_HL.wiff 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 5 10 15 20 25 30 620.67 186.16 Q8WUC1 Q96RF8 TUBULIN, BETAHomo sapiens (Human). SSA1.- 5.- Homo sapiens (Human). C25437 BAB27292 beta-3 chain - NID: - Mus musculus tubulin AK010960 mouse CAC39526 K2C1_HUMAN SEQUENCE Keratin, type II cytoskeletal 1 (Cytokeratin 1) 15 FROM PATENT WO0129232.-HUman AAH19046 A45935 SIMILAR TO IMMUNOGLOBULIN HEAVY hsc70 - mouse dnaK-type molecular chaperone CONSTANT GAMMA 3 K1CJ_HUMAN KRHU0 Keratin, type 10, type I, cytoskeletal - human keratin I cytoskeletal 10 (Cytokeratin 10-(Human). I37383 C25437 soluble protein - human - mouse FAS tubulin beta-3 chain A24903 Q9CWA2 alpha-1 chain - Chinese TRANSPORTING MITOCHONDRIAL F1 tubulin ATP SYNTHASE, H+ hamster A44861 CAA30026 67K type II epidermal - human keratin, HSHA44G NID: - Homo sapiens I38707 CAB59134 Fas ligand - SEQUENCE 1 FROM PATENT WO9818921 PRECURSOR human AAG41947 A44861 AF304164 NID: 67K type II epidermal - human keratin, - Homo sapiens Q9BDN1 Q9BWB7 CD95L PROTEIN.- CercocebusPROTEIN 9B (MORTALIN-) (Human). HEAT SHOCK 70KD torquatus atys CAA82315 HSKERAT9 NID: - Homo sapiens A26168 ribophorin I precursor - human AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN (Human). PT0207 Ig gamma chain C region - chimpanzee NUCL_HUMAN PWHUA Nucleolin (Protein C23).- (Human). H+-transporting two-sector ATPase alpha chain precursor - human 1ATS JC1473 shock cognate protein 70 kD (44 kD chaperone ATPase chain - mouse heat H+-transporting ATP synthase (EC 3.6.1.34) alpha 1FC1A I77403gamma-1 chain C region (Fc- fragment), chain A - human Ig tubulin alpha-1 chain human I61769 AAA57233 6d, type II - human (fragment) keratin MUSHP7A2 NID: - Mus musculus A29904 CAA82315 5, type II, epidermal - -human sapiens keratin HSKERAT9 NID: Homo A40389 A29904 translation elongation factor eEF-1 alpha chain (clone pS1) - rat keratin 5, type II, epidermal - human HHHU84 75 heat shock protein95 100[validated] - human HSU8621480 NID: 85 - Homo sapiens 90-beta 105 110 115 35 40 45 50 55 AAB46730 60 65 70 90 Time, min AAB86467 CAD23746 IMMUNOGLOBULIN GAMMA HEAVY CHAIN CONSTANT REGION IMMUNOGLOBULIN GAMMA HEAVY CHAIN .-(Human). Q10466 B26168 HEART ISOFORM N2-B (EC human (CONNECTIN).-(Human). TITIN, ribophorin II precursor - 2.7.1.-) Q8WZ42 CAA34756 Homo sapiens (Human). sapiens Max. 4.0e5 cps. TITIN.HSEF1AC NID: - Homo 1D3OA S21097 trypsin (EC 3.4.21.4), chain Aprecursor - bovine alpha-1-antitrypsin - pig CAC20457 S04652 603.33 IMMUNOGLOBULIN HEAVY CHAIN(EC 3.6.1.38) GAMMA 4.- (Human). Ca2+-transporting ATPase CONSTANT 2, - pig CAA41735SHOCK PROTEIN - Bos taurus HEAT BTBSA NID: 70 TESTIS VARIANT.- (Human). 655.82 O75634 Q8WTZ6 Q9UK02 RIBOSOMAL PROTEIN (FRAGMENT).- Homo(Human). (Human). BIP L18.- Homo sapiens sapiens 1NBMC KRHUEA f1-atpase (EC 3.6.1.34) delta -and 1 epsilon subunits, chain C - bovine keratin 6a, type II human Q96PE2 A22224 TUMOR ENDOTHELIAL MARKER 4.- Homo sapiens (Human). actin alpha, vascular smooth muscle - mouse Q9R1Q3 Q96GA6 GLIAL FIBRILLARY ACIDIC PROTEINMGC:15420).- Homo sapiens(Rat). UNKNOWN (PROTEIN FOR ALPHA.- Rattus norvegicus (Human). 328.19 ITSH Q8WZ42 alpha-1-antitrypsin Homo sapiens (Human). TITIN.- precursor - sheep CAA27396 130.11 I84741 MMACTBR2 helicase - mouse RNA NID: - Mus musculus Q8WXH0 Q9TS10 NUANCE.- HomoAPAMIN BINDING PROTEIN.- Bos taurus (Bovine). 78 KDA sapiens (Human). 405.33 Q9GL40 AAH02690 FAS ANTIGEN.- Macaca mulatta (Rhesus macaque). BC002690 NID: - Homo sapiens 337.17 A33370 I48385 H+-transporting ATP synthasemousechain precursor, mitochondrial RNA helicase TNZ2 - beta 864.45 CAB76567 Q96FZ6 MMU250841 NID: - Mus musculus HEAT SHOCK 60KD PROTEIN 1 (CHAPERONIN).(Human). 171.17 582.29 Q9GK28 AAA56753 FAS ANTIGEN APO-1/CD95.-Homo sapiens HSU15637 NID: - Macaca arctoides (Stump-tailed macaque). Q9BZL4 CAA58470 MYOSIN BINDING SUBUNIT -85.- Homo sapiens (Human). HSPXMP11 NID: Homo sapiens 643.87 603.69 ………………………………… JQ0028 cytokeratin 19 – mouse ………………………………… 158.06 600 spectra 680 spectra 120.11 171.18 449.15 607.38 35 40 45 50 55 60 65 Time, min 70 75 80 85 90 95 100 105 110 115 Slide 64: FILTERED RESULTS Database search Database search Database search 1. 2. 3. 4. 1. 2. 3. 4. 1. 2. 3. 4. Protein 1 Protein 2 Protein 3 … Protein 1 Protein 2 Protein 3 … Protein 1 Protein 2 Protein 3 … Total list sample 1 1. 2. 3. 4. 5. 6. 7. Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Protein 6 … Total list sample 1 1. 2. 3. 4. 5. 6. 7. Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Protein 6 … Total list sample 2 1. 2. 3. 4. 5. 6. 7. Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Protein 6 … Subtract from each list : 1) 1) 1) COMMON CONTAMINANTS ( PROTEINS STICKING TO BEADS) LIGAND-COPURIFYING PROTEINS (EX SJOGREN SYNDROME 52 KDA) COMMON HITS (WHAT IS IN BOTH LISTS) Slide 65: FILTERED RESULTS MASCOT DATABASE SEARCH Fas (CD95) complex ICE8_HUMAN (Q14790) Caspase-8 MASCOT DATABASE SEARCH BAFF receptor complex RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen RS3_HUMAN FADD_HUMAN (P23396) 40S ribosomal protein S3 (Q13158) FADD RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen RS3_HUMAN (P23396) 40S ribosomal protein S3 K2C1_HUMAN GC1_HUMAN CFLA_HUMAN regulator RS8_HUMAN (P04264) Keratin (P01857) Ig gamma-1 chain C region (O15519) CASP8 and FADD-like apoptosis K2C1_HUMAN GC1_HUMAN (P04264) Keratin (P01857) Ig gamma-1 chain C region (P09058) 40S ribosomal protein S8 GBLP_HUMAN protein TNR6_HUMAN (P25388) Guanine nucleotide-binding (P25445) TNF-family receptor CD95 GBLP_HUMAN protein (P25388) Guanine nucleotide-binding RL7A_MOUSE RL7_HUMAN ICEA_HUMAN (P12970) 60S ribosomal protein L7a (P18124) 60S ribosomal protein L7 (Q92851) Caspase-10 RL7A_MOUSE RL7_HUMAN (P12970) 60S ribosomal protein L7a (P18124) 60S ribosomal protein L7 PHB_HUMAN (P35232) Prohibitin K22E_HUMAN (P35908) Keratin K22E_HUMAN (P35908) Keratin Slide 66: • One major evolution of proteomics technologies in the last years has been the introduction of gel-free approaches for large scale protein identification and quantification • These methods combine isotope labelling, separation techniques and mass spectrometry Slide 67: WORKFLOWS 3: isotope labelling strategies Slide 68: Relative quantification : Comparison of proteins from samples A vs B ? Which proteins change in amount and how much ? Applications : -Healthy vs. diseased tissues -Healthy vs. diseased body fluids -Drug treated / untreated cells -Stimulated / unstimulated cells -Mutants / wt cells -…….. Slide 69: Relative quantitation : stable isotope labelling is very fashionable! Sample A : light isotope mix, digest Quantitate and identify ( MS) +TOF MS: Experiment 1, 44.071 to 46.012 min from 181203_QS_MQ_RuedaICAT1_long... a=3.56275471721098790e-004, t0=7.24150134716619500e+001 8.96 320 300 280 260 240 220 In te n s ity , c o u n ts 200 180 160 140 120 100 80 60 40 20 0 930 940 950 960 2.00 970 m/z, amu 980 990 1000 1010 1020 1.00 10.96 Max. 649.4 counts. Sample B : heavy isotope ∆m = 9 Da Peptide from sample A 968.52 9.96 Peptide from sample B Slide 70: How to label ? -chemically, post protein synthesis  “specific” chemical modification of AA side chain (+) any sample can be done (-) side reactions -metabolically, during protein synthesis Incorporation of one or more labelled amino acid (+) “native” proteins (-) need cultivable organism Slide 71: Isotope Coded Affinity Tag (ICAT) reagents State 1 [protein1] [protein2] . . [proteinn] Transition states State 2 [protein1] [protein2] . . [proteinn] O N S Biotin tag N O N XX O XX O XX O O XX N I d0- or d8-ICAT (X= H or D) Linker (heavy or light) Thiol reactive Slide 72: New Methods : ICAT:quantitation and identification Cell State 1 Cell State 2 Modify with (H8)-ICAT HH HH Biotin HH HH N H O I Combine samples Modify with (d8)-ICAT DD DD Biotin DD DD N H O I HS•Digest Trypsin •Purify Cys-containing peptides on avidin column -SH Intensity Identify proteins by MS/MS aa4 Intensity aa2 aa1 A1 B1 A2 A3 B2 aa3 m/z m/z Quantitate protein levels by H8 / D8 peak heigth ratios Slide 73: Pair wise ICAT with Multidimensional Chromatography 1) 2) control treatment A) Identify 100 y4 y5 y6 y7 y8 y9 y10 y11 y12 y13 y14 y15 y16 y17 R.A. (%) d0 ICAT label d8 D 50 I NN b2 b3 b4 b5 T b6 I b7 QS b8 b9 LTADA b10 b11 b12 b13 b14 b15 d0/d8 d0 AD AT LS QI T N N ID OD214 3) 0.5 0.4 0.3 0.2 0.1 0.0 Combine & proteolyze Ion-Exchange 0 0.6 400 800 1200 1600 2000 KCl [M] 0.3 0.0 10 6) d0 B) Quantify Area = 1.21x109 m/z 4) 100 Fraction # (Time (min)) 20 30 40 50 60 70 Avidin Affinity Chromatography LC-MS/MS 100 R.A. (%) 5) Area = 1.01x109 % AcN % AcN 50 50 d8 d8/d0 = 1:0.83 d8/ 0 0 10 20 30 40 50 60 70 Time (min) Slide 74: ICAT (+) and (-) - relative protein quantification by MS + - simplification of complex mixtures by selecting a subset of peptides after digestion - eliminate analytical variability by mixing samples - protein quantification unreliable for weak signals - - affinity purification (avidin) : losses for low amounts - multiple side reactions possible ~15 different isotope labelling methods developed in the last 5 years !! Slide 75: Recent ICAT studies (R. Aebersold’s group) Wollscheid B, von Haller PD, Yi E, Donohoe S, Vaughn K, Keller A, Nesvizhskii AI, Eng J, Li XJ, Goodlett DR, Aebersold R, Watts JD. Lipid raft proteins and their identification in T lymphocytes. Subcell Biochem. 2004;37:121-52 Yan W, Lee H, Yi EC, Reiss D, Shannon P, Kwieciszewski BK, Coito C, Li XJ, Keller A, Eng J, Galitski T, Goodlett DR, Aebersold R, Katze MG. System-based proteomic analysis of the interferon response in human liver cells. Genome Biol. 2004;5(8):R54. Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A, Wijgers N, Jaspers NG, Raams A, Argentini M, van der Spek PJ, Botta E, Stefanini M, Egly JM, Aebersold R, Hoeijmakers JH, Vermeulen W. A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A. Nat Genet. 2004 Jul;36(7):714-9. Ranish JA, Hahn S, Lu Y, Yi EC, Li XJ, Eng J, Aebersold R. Identification of TFB5, a new component of general transcription and DNA repair factor IIH. Nat Genet. 2004 Jul;36(7):707-13. Hardwidge PR, Rodriguez-Escudero I, Goode D, Donohoe S, Eng J, Goodlett DR, Aebersold R, Finlay BB Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli. J Biol Chem. 2004 May 7;279(19):20127-36. Zhang J, Goodlett DR, Peskind ER, Quinn JF, Zhou Y, Wang Q, Pan C, Yi E, Eng J, Aebersold RH, Montine TJ. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid. Neurobiol Aging. 2005 Feb;26(2):207-27. Marelli M, Smith JJ, Jung S, Yi E, Nesvizhskii AI, Christmas RH, Saleem RA, Tam YY, Fagarasanu A, Goodlett DR, Aebersold R, Rachubinski RA, Aitchison JD. Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane. J Cell Biol. 2004 Dec 20;167(6):1099-112. Epub 2004 Dec 13. Slide 76: SILAC Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002 May;1(5):376-86. • Label light / heavy cultures (Leu d0 / d3) • Stimulate heavy cells • Mix cells or lysates • Purify fraction of interest • Analyse by LC-MS/MS (->ID) • Quantify signals of ion pairs Slide 77: SILAC (+) and (-) • relative protein quantification by MS • eliminate praparative variability by mixing samples immediately after culture • eliminate analytical variability • peptides in native state (no side reactions) • protein quantification unreliable for very weak signals + - • mass shift variable (dependent on number of residues) • only feasible with organisms in culture Slide 78: Recent SILAC articles Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002 May;1(5):376-86. Blagoev B, Ong SE, Kratchmarova I, Mann M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol. 2004 Sep;22(9):1139-45. Epub 2004 Aug 15. Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faergeman NJ, Mann M, Jensen ON. Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway. Mol Cell Proteomics. 2005 Mar;4(3):310-327. de Hoog CL, Foster LJ, Mann M. RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers. Cell. 2004 May 28;117(5):649-62. Ong SE, Kratchmarova I, Mann M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J Proteome Res. 2003 Mar-Apr;2(2):173-81. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol. 2003 Mar;21(3):315-8. Epub 2003 Feb 10. Foster LJ, De Hoog CL, Mann M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A. 2003 May 13;100(10):5813-8. Epub 2003 Apr 30. Slide 79: Other fields • Proteome subsets – Phosphoproteome – Ubiquitinated proteins –… • Clinical proteomics (marker discovery) – Too vast to summarise • Proteome imaging – MALDI of tissues