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Mass Spectrometry and Proteomics - Application of Proteomics Research for New Drug Targets Study

Mass Spectrometry and Proteomics - Application of Proteomics Research for New Drug Targets Study. Yan-Hui Liu Structural Chemistry/Mass Spectrometry Schering-Plough Research Institute. Protein ID by MS in Combination with Various Protein Fractionation/Separation Methods. 2 DE-MS

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Mass Spectrometry and Proteomics - Application of Proteomics Research for New Drug Targets Study

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  1. Mass Spectrometry and Proteomics - Application of Proteomics Research for New Drug Targets Study Yan-Hui Liu Structural Chemistry/Mass Spectrometry Schering-Plough Research Institute

  2. Protein ID by MS in Combination with Various Protein Fractionation/Separation Methods • 2DE-MS • ability to observe global changes in the total cellular protein complement and post-translational modifications associated with differential gene expression or compound treatments. • specific classes of proteins may be absent or under-represented: very acidic or basic proteins, extremely small or large proteins, membrane proteins, and low abundance proteins. • Multi-Dimensional Liquid Chromatography (MDLC)-MS: • SCX/RP-HPLC of enzymatically digested total cell lysate • signature peptide approach: Isotope-Coded Affinity Labeling • protein pre-fractionation prior to 1, 2-DE or MDLC • Immunoprecipitation or Affinity Pre-enrichment Combined with 1, 2-DE, or MDLC-MS • identify biomarkers and binding partners of target protein(s)

  3. Functional Study of Drug Targets Using Proteomics and Mass Spectrometric Tools • Analysis of Protein Expression -- Expression Proteomics • strategies • identification of the function of antibacterial target Unk4 (S. aureus) through 2-D gel electrophoresis followed by mass spectrometric protein identification • Analysis of Protein Function -- Functional Proteomics • functional study of novel drug target by identification and characterization of its natural ligand • Analysis of Protein Structure/Protein Complexes -- Chemical Cross-linking/Mass Spectrometry

  4. Peptide Mass Fingerprinting • MALDI-MS • Comparing experimental PMF with theoretical PMF from protein databank • Facilitates rapid protein identification from simple mixtures (e.g. 2D PAGE) by MALDI MS

  5. MS/MS and dB searching • Using fragmentation model to compare experimental MS/MS spectra with theoretical MS/MS spectra generated from the in silico digestion of known proteins. • Facilitates rapid protein identification by matching experimentally characterised peptides to databank proteins. Courtesy of Micromass, Ltd. (UK)

  6. 2-D Spot Identification by PMF Zoomed view after automated spot picking SYPRO Ruby stained E.coli gel MW (kDa) 7988.7 100 1 90 80 70 2 60 % Intensity 50 40 30 Spot #1: unknown protein from gene y 20 10 14.4 20.1 30.0 97.0 66.0 45.0 0 0 499.0 1999.4 3499.8 5000.2 6500.6 8001.0 Mass (m/z) pI 4.0 linear IPG pI 7.0

  7. Protein identification of RP-HPLC peak 2: non-significant identification of rAd pX by peptide mass fingerprint 3037.78 3053.95 819.4595 # 30000 CHCA 3069.08 25000 20000 443.2141 # 1088.6475 # 563.2941 15000 459.2046 # Counts 2547.2346 628.2894 1123.5795 Trypsin 923.4686 1020.5098 1993.9854 2273.1509 1671.8293 10000 5000 0 500 1000 1500 2000 2500 Mass (m/z) Rank MOWSE Protein Species SwissProt Protein Name Score MW (Da)/pI Accession # 1 77.6 37832.2/8.23 CHVP1 Q84424 MRNA Capping Enzyme 2 57.2 48778.4/8.39 HAEIN P44856 NADH Dehydrogenase 3 48.9 59428.2/7.07 ORENI P70091 Cytochrome P450 19A133 3# 48.7 8845.7/12.88 ADE02 P14269 Late L2 MU Core Protein Precursor (11 KD Core Protein) (Protein X) 4 45.4 23123.2/9.06 BRUCA Q45110 25KD Outer-Membrane Immunogenic Protein Precursor MS-Fit Search

  8. y4-NH3 [459.78] MH+ 819.4555: PSD y7 y6 y5 y4 y3 y2 y1 5000 F P V P G F R b2 b3 4000 y4 [476.94] 3000 Counts V [72.01] y7-NH3 [801.54] y1-NH3[158.13] 2000 y6 [672.83] y2-NH3 [305.62] y3-NH3 [362.23] b2 [245.25] PGF[302.61] GF [205.53] y5-NH3 [558.45] y6-NH3 [655.78] y5 [575.31] a2 [217.48] b3 [344.42] P/R[70.00] 728.43 y1 [174.97] y2 [322.53] 1000 777.73 R [111.97] R [119.83] 618.29 0 100 200 300 400 500 600 700 800 Mass (m/z) Obtain sequence tag of tryptic peptide (m/z 819.5) by MALDI/PSD

  9. Rank # Sequence MH+ MH+ Protein Species SwissProt Protein Unmatched Caclc. Error MW (Da) Accession Name Ions (Da) (Da) /pI # 1 16/30 (R)FPVPGFR(G) 819.4517 0.0038 8845.7/12.88 ADE02 P14269 Late L2 MU Core Protein Precursor (11 KD Core Protein) ( Protein X) 2 20/30 (K)VPFFPGR(G) 819.4517 0.0038 82989.3/5.06 BACST P14412 Peroxidase/ Catalase ox NovelAdenovirus Protease Cleavage Site of pX (?) -------------------------------propeptide----------------------------------------------Late L2 MU Core Protein-- MALTCRLRFPVPGFRGRMHR RRGMAGHGLT GG MRRAHHRR RRASHRRMRG -----------------------------propeptide------------------------ G ILPLLIPLI AAAIGAVPGI ASVALQAQRH Ad protease (?) * Substrate specificity of Ad protease: (M,I,L)XGG X or (M,I,L)XGX G MS-Tag search against SwissProt identify the rAd pX

  10. Proteins of unknown function 14% bacterial broad spectrum targets 82% gram positive only 45% gram negative only 30% fungal targets Access function by expression proteomics identify global protein expression patterns associated with conditional gene expression or chemical perturbations identify potential function/pathway for unknown targets develop novel biochemical assays based on identification of protein function derived from pathway analysis Identification of the Function of Unk4 for Antibacterial Drug Discovery via Expression Proteomics

  11. Experimental Conditions

  12. Growth Curves

  13. Growth Curves

  14. T=0, 3 (+IPTG) Cy3 image T=0, 3 (-IPTG) Cy5 image Proteomic T=0 Results

  15. T=3, 2(+IPTG) Cy5 image T=3,2(-IPTG) Cy3 image Response Effect of Unk4 Knockout (proteomic T=3 results) up-regulated down-regulated

  16. Review dB Search Results assigned sequence of the peptide based on the MS/MS data Protein ID

  17. Identified on Average Spot "normal" and Response to Volume Change gene Identified Proteins shutoff gels unk4 shutoff (8 gels) dihydroxy-acid dehydratase no increases 12.57 acetolactate synthase (large subunit) no increases 14.61 1-pyrroline-5-carboxylate dehydrogenase yes decreases 3.16 1)formyltetrahydrofolate synthetase yes decreases 3.57 2)no hit-hypothetical protein no decreases 3.57 chorismate mutase homolog no increases 4.38 1)threonine dehydratase/deaminase no increases 7.59 2)conserved hypothetical protein no increases 7.59 ketol-acid reductoisomerase (2 spots-8 gels) no increases 10.52/10.46 nucleoside diphospate kinase yes decreases 3.77 1)xx no decreases 9.25 2A)copper transporting ATPase no decreases 9.25 2B)hypothetical protein no decreases 9.25 phosphoribosylformylglycinamidine synthase I no decreases Unk4 shutoff Protein IDs ilvD ilvB ilvA ilvC

  18. K-means clustering 15 ilvB* -23.7 10 ilvD* -21.9 leuA -20.6 5 leuB -18.4 0 leuC -18.4 -5 -10 ilvA* -11.0 ilvC* -15.9 -15 -20 unk4 +7.7 x nc AAB v AAC AAB v AAD AAB v AAE AAB v AAF AAB v AAG AAB v AAH + - + - + - 1h 2h 3h Expression Level Clustering After Unk4 Shutoff Absolute Differential Expression (3h) Expression Increasing *consistent with expression proteomic results

  19. Valine, Leucine and Isoleucine Biosynthesis Threonine deaminase Acetolactate synthase Acetolactate synthase Ketol-acid reductoisomerase Ketol-acid reductoisomerase Ketol-acid reductoisomerase Ketol-acid reductoisomerase Dihydroxy-acid dehydratase Dihydroxy-acid dehydratase

  20. S. aureus Organization Valine, Leucine and Isoluecine Biosynthesis Regions x y z u v ilvD ilvB ilvN ilvC leuA leuB leuC ilvA leuD x= unknown protein y = unknown protein z = unknown protein u = unknown protein ilvD = dihydroxy-acid dehydratase (20566-22281) ilvB = acetolactate synthase (large subunit) (22282-24078) ilvN = acetolactate synthase (small subunit) (24063-24332) ilvC = ketol-acid reductoisomerase (24394-25473) leuA = isopropylmalate synthase (25479-27032) leuB = 3-isopropylmalate dehydrogenase (27029-28081) leuC = isopropylmalate dehydratase subunit (28095-29465) leuD = isopropylmalate dehydratase subunit (29566-29937) ilvA = theonine dehydratase/deaminase (30031-31335)

  21. Unk4 Hypothesis • Because an Unk4 depletion causes an up-regulation of an amino acid biosynthesis pathway -- it may affect a transporter/transporter component, either by processing or allowing secretion. This transporter could allow entry of amino acid or peptide fragments into S. aureus. • Testing the hypothesis • confirm the 2-D DIGE expression proteomics results by Isotope-Coded Affinity Tags (ICAT)/Multi-Dimensional Liquid Chromatography (MDLC)/MS approach. • identify Unk4 interacting partners through MS studies of protein complexes.

  22. Protocol for Quantification by ICAT/MDLC/MS Approach Control Experimental Reduce Reduce Trypsin digest Trypsin digest ICAT (1H) ICAT (2H) Mix samples Affinity select RPC or CE MALDI or ESI Bioinformatics

  23. control CH-CH2-S-CH2-CO-NH Linker arm (1H8) biotin Trypsin digestion Note the deuterium label experimental CH-CH2-S-CH2-CO-NH Linker arm (2H8) biotin 1H8-ICAT labeled peptide x 2H8-ICAT labeled peptide x intensity m/z The ICAT Approach R. Aebersold Anal. Chem. January 2000.

  24. Frequency of Low Abundance Amino Acids and Post-Translational Modification (E. coli) Amino acid/PTM Av. number per protein tyrosine 3.5 cysteine 2.8 histidine 2.1 methionine 1.7 tryptophan 1.1 glycosylation 0-5 phosphorylation 0-5

  25. Rat spinal cord (100 ng) RP-HPLC C18 column Cation exchange column (SP/8HR) Anion exchange column (MonoQ) DEAE column RP-HPLC C18 column Fractions for MS structural determination Purification of SP1999 Ligand(s)

  26. 100 LC/ESI MS of Purified Ligand of SP1999 227.0 - [(TFA) -H] 2 % 249.0 5’-ADP - [(TFA) -2H+Na] 2 426.1 (ADP-H) - 346.1 - (AMP-H) 0 m/z 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 159 79 N H 2 N N O O 134 LC/ESI-MS-MS of m/z 426 100 N P O P O N H O O 134 O H O H 159 - H P O O H O H 3 -Base - (HPO -H) 3 % - H O 346 2 79 - (M-H) 291 - (M-H PO -H) 3 4 408 - H O 426 2 328 - (M-H O-H) 273 2 408 - H P O 3 273 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 328 MS Identification of Novel Ligand for SP1999 MS Identification of Novel Ligand for SP1999 (C10H13O9N5P2: high resolution MS)

  27. Summary • Protein Identification • Integrated MS techniques (MALDI MS and ESI Q-TOF MS/MS) with 1-DE, 2-DE, chromatographic separation methods, and bioinformatics tools for identification of proteins for drug targets studies and target validation. • Protein Functional Study • Identified SP1999 as a novel P2Y (G-linked) receptor for ADP. This result allowed the functional hypothesis to be generated and confirmed, which led to the validation of SP1999 as a viable target for anti-throbotic therapy. • Protein Structural Study • The information of inter-lysine distances and solvent accessibility of lysine residues of hIL-10 derived from Chemistry Cross-linking/Mass Spectrometry study matched very well with those obtained from crystallography study. • This method will provide valuable distance-constraint and amino acid solvent accessibility information for protein structural modeling using bioinformatics tools. It can also be applied for protein-protein interaction studies.

  28. Acknowledgement Leigh Ann Giebelhaus Todd Black Fang L. Zhang Frederick J. Monsma, Jr. Nicholas J. Murgolo Wei Ding Gary Vellekamp David Wylie Fred Poeter Shihong Wang Guodong Chen James Pai Birendra N. Pramanik John Piwinski

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