12/6/12. Dr. Sanjeeva Srivastava. IIT Bombay 2

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1 Dr. Sanjeeva Srivastava IIT Bombay Case studies 2DE: Drug treatment in malaria parasite Plasma proteome analysis of SARS An overview of DIGE technique Case study DIGE: Serum proteome analysis of prostate cancer IIT Bombay 2 1

2 Case study-1 Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria. Proteomics. 2005, 5, 1849 IIT Bombay CoArtem is a combination of artemisininderivative artemether with lumefantrine Drug of choice for all cases of non-severe malaria worldwide Artemisinin drug action is mediated specifically through its endoperoxide moiety IIT Bombay 4 2

3 Plasmodium falciparum Proteome IIT Bombay 5 Late ring stage of Plasmodium falciparum Plasmodium falciparum Culture of late ring stage pf Stationary Phase Log Death Phase Log Phase Lag Phase Time Microscopic field showing Pf in its late ring stage in the synchronised culture IIT Bombay 6 3

4 Fractionation of the P. falciparum proteome Synchronised parasites isolated from host erythrocytes Washed and solubilized in a Tris buffer Tris-insoluble fraction was subjected to two further extractions in urea based lysis buffers IIT Bombay 7 _ Protein Samples loaded into sample wells Sample wells + Glass/ Plastic plates Gel Buffer Protein bands after staining IIT Bombay 8 4

5 CoArtem = Lumifantrine + Artemether Reaction with Late ring stage of Plasmodium falciparum Glycolytic Enzymes Alterations in Parasite Proteome IIT Bombay 9 Membrane associated calcium binding protein Aspartic proteinase (HAP) HSP60, 70, 90 Enolase Fructose biphosphate aldolase Phophoglycerate kinase Artemether D D D Lumefantrine IIT Bombay 10 5

6 Stress Response Proteins Glycolytic Enzymes ARM LM ARM LM pregulated Downregulated IIT Bombay 11 Investigation of alterations to the parasites proteome induced by the two components of CoArtem, artemether and lumefantrine Established relationship between pharmacologically relevant concentration and time of exposure for the two components of CoArtem IIT Bombay 12 6

7 Case study-2 Chen et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry. Proc Natl Acad Sci SA. 2004, 101, IIT Bombay Plasma proteome analysis of severe acute respiratory syndrome (SARS) Technique: 2DE and Mass Spectrometry IIT Bombay 14 7

8 Buffer 12/6/12 Membrane Glycoprotein (M) Spike Glycoprotein (S) Respiratory Disease Droplets of saliva or mucus infected with SARS virus Nucleocapsid Phosphoprotein (N) RNA SARS Virus IIT Bombay 15 4 SARS patients 22 plasma samples 6 Healthy Patients 6 Healthy Controls 385 Proteins with >2 fold change excised _ Sample wells Protein Samples loaded into sample wells Protein bands after staining + Gel Glass/ Plastic plates 38 Spots selected for Protein Identification Trypsin Trypsin Digestion IIT Bombay 16 8

9 IIT Bombay 17 Mass Analyzer #1 Collision Cell Mass Analyzer #2 Ion Source Intensity LC-MS Data Analysis Your name Proteomics Search title Database (s) SwissProt NCBInr MSDB Taxonomy Bacterial Fixed modification s Variable modification Sample protein Oxidation (M) [email protected] Enzyme Trypsin Quantitation Peptide tol. Peptide charge Data file Data format Instrument 1.2 Da # C 13 MS/MS tol. 0.2 Da Monoisotopic Average Choose file Precursor Elution time (min) ESI-Q-TOF Start search IIT Bombay 18 9

10 Differentially expressed proteins IIT Bombay 19 SDS-PAGE PVDF IIT Bombay 20 10

11 Reducing Recomb PrxII Normal Patient Sample 1 Patient Sample Non reducing Conditions Recomb PrxII Normal Patient Sample 1 Patient Sample kda 22 kda IIT Bombay 21 Peroxiredoxin II Virus HLA Class I Viral peptide T cell CD8 Receptor T cell IIT Bombay 22 11

12 SARS Corona Virus Infection Acute Phase Proteins in Excess Indicates bad liver responsible for weak defense against SARS IIT Bombay 23 12

13 Sample 1 Cy3 Cy3 Excitation Protein Samples Standard (1+2) Sample 2 Cy2 Cy5 Cyanine dyes Mix Cy2 Excitation Cy5 Excitation IIT Bombay 25 Sample mixing and 2-DE Sample 1 Sample 2 Internal standard ph 3 Increasing ph Direction of migration, Direction of migration, ph 9 IIT Bombay 26 13

14 DIA Analysis ph 4 ph 7 Gel Viewing Cy3 Excita5on DeCyder Software ph 4 ph 7 Cy2 Excita5on ph 4 Increasing pi ph 7 Cy5 Excita5on IIT Bombay DeCyder Software HC PC Representative Three dimensional Protein expression profiles 28 IIT Bombay 14

15 LC MS/MS Mass Analyzer #1 Collision Cell Mass Analyzer #2 Ion Source LC- MS Data Analysis Your name Search -tle Proteomics Sample protein Intensity Database(s) Taxonomy Fixed modifica-ons Variable modifica-on SwissProt NCBInr MSDB Bacterial Oxida-on (M) Enzyme Trypsin Quan-ta-on Elu-on -me (min) Mass Spectra - PC Pep-de tol. Pep-de charge Data file Data format Instrument 1.2 Da # C 13 MS/MS tol. 0.2 Da Choose file ESI- Q- TOF Monoisotopic Precursor Start search Average Mass Spectra - HC IIT Bombay 15

16 Case study-3 Byrne et al. 2D-DIGE as a Strategy To Identify Serum Markers for the Progression of Prostate Cancer J Proteome Research. 2009, 8, IIT Bombay Identification of serum markers depicting progression of prostate cancer using DIGE Prostrate Cancer Serum Healthy Control Serum Healthy Human PSA Prostrate Cancer DRE Biopsy Tissue Sample Proteomic Analysis Tissue Sample Diagnostic Markers for early prostrate cancer IIT Bombay 16

17 Mobile phase reservoir (salt solu5on) Injec5on of Sample Size Separa5on Elu5on of large solutes Elu5on of small solutes Loading of unpurified Protein Mixture Sample Ligand solu5on Deriva5zed sta5onary phase Effluent sample frac5ons Collected Concentra5on Detector IIT Bombay 2 Cy3 Standard (1+2) 1 Cy2 Cy5 Mix Cyanine dyes IIT Bombay 17

18 Depleted Cancer Serum (cohort 1) Depleted Cancer Serum (cohort 2) _ Sample wells Protein bands after staining- PC 2- D Electrophoresis + Buffer Gel Glass/ Plastic plates SDS-Polyacrylamide Gel IIT Bombay IIT Bombay 18

19 Mass Analyzer #1 Collision Cell Mass Analyzer #2 Ion Source Intensity LC-MS Data Analysis Your name Proteomics Search title Database (s) SwissProt NCBInr MSDB Taxonomy Bacterial Fixed modification s Variable modification Sample protein Oxidation (M) [email protected] Enzyme Trypsin Quantitation Peptide tol. Peptide charge Data file Data format Instrument 1.2 Da # C 13 MS/MS tol. 0.2 Da Monoisotopic Average Choose file Precursor Elution time (min) ESI-Q-TOF Start search IIT Bombay 37 DeCyder Progenesis Pigment epithelium-derived factor Zinc-α2-glycoprotein Ficolin 3 Apolipoprotein A-II D D D D IIT Bombay 38 19

20 2ry Antibody 1 0 Antibody PEDF Target protein Capture Ab 96 well plate ZAG ELISA IIT Bombay Chromogen Serum Blocking reagent Tissue IIT Bombay 20

21 Serum markers reflective of pathological grade and stage are beneficial for identification of appropriate treatment strategies Confirmed differential expression of PEDF and ZAG by various validation techniques PEDF a potential marker of early stage prostate cancer prediction IIT Bombay 41 Two dimensional electrophoresis (2DE and 2D- DIGE) can be applied for various applications Case studies Drug treatment in malaria parasite Plasma proteome analysis of SARS Serum proteome analysis of prostate cancer IIT Bombay 42 21

22 Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria. Proteomics. 2005, 5, 1849 Chen et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry. Proc Natl Acad Sci SA. 2004, 101, Byrne et al. 2D-DIGE as a Strategy To Identify Serum Markers for the Progression of Prostate Cancer. J Proteome Research. 2009, 8, Issaq H, Veenstra T. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): advances and perspectives. Biotechniques. 2008, 44, 697-8, 700. IIT Bombay John F. Timms, Rainer Cramer Professor, PROTEOMICS. Difference gel electrophoresis. Volume 8, Issue 23-24, pages , No December Ray et al. Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response. Journal of Proteomics. Volume 75, Issue 10, 6 June 2012, Pages IIT Bombay 22