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Yeast Proteome Chip

Yeast Proteome Chip. Global Analysis of Protein Activities Using Proteome Chips. Snyder Lab Zhu, Bilgin, Bangham, Hall, Casamayor, Bertone, Bidlingmeier, Snyder . Why Develop Protein Microarray-Chip Technology?. DNA microarrays Gene expression analysis Genotyping Toxicogenomics

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Yeast Proteome Chip

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  1. Yeast Proteome Chip Global Analysis of Protein Activities Using Proteome Chips Snyder Lab Zhu, Bilgin, Bangham, Hall, Casamayor, Bertone, Bidlingmeier, Snyder

  2. Why Develop Protein Microarray-Chip Technology?

  3. DNA microarrays Gene expression analysis Genotyping Toxicogenomics Pharmacogenomics Diagnostics Protein microarrays Protein expression analysis Drug discovery Clinical diagnostics Others emerging Applications of Biochips

  4. Yeast Proteome Chip • First Protein “chocolate” Chip $ 2.99

  5. Yeast Proteome ChipBuilding up the yeast ORF collection Aimed at cloning 6144 yeast ORFs • 5871 PCR amplified ORFs cloned into pEKGH • 89 % with correct ORF ID and “in frame” expression clones • 300 represented >1 copies To complete the collection: • ~300 new unique clones sent for sequence confirmation • ~950 low quality sequencing

  6. Experimental Approach

  7. Chip Fabrication, Probing and Detection :Technical Issues • High-throughput fusion protein purification • Printing chips Suitable surface chemistry for attachment of proteins and retaining integrity, orientation, structure, activity Cross-contamination, Spot size, comets etc. • Detection Sensitive-specific probe with Retain signal during washing Low background, high signal/noise

  8. Design of protein chips Chip probed with -GST antibody and signals detected after Cy5-conjugated IgG 12,938 data points Each spot corresponds to ~30 fg-~50 pg protein

  9. Analysis of the Yeast Proteome Chip • Protein –Protein interactions • 1° Ab against target protein domain • 1° Ab against interacting partner protein • Biotin labeled protein detected by Cy3 conjugated streptavidin • Protein-Nucleic acid interactions • Cy3 labeled genomic DNA • Cy3 labeled mRNA • Protein-Lipid interactions • Biotin-conjugated liposome-phopshotidyl phosphate detected by Cy3 conjugated streptavidin

  10. Detection of different interactions on yeast proteome chips PI(3,4,5)P3 PC Calmodulin Genomic DNA

  11. Results : Protein- Protein InteractionsCalmodulin Known interactions (4/8): • Cmk1p, Cmk2p type I, type II calcium/calmodulin-dependent serine/threonine kinases • Cmp2 (Cna2p) calcineurin • Arc35 actin-organizing complex, endocytosis 33 other potential in vitro interactors: Rpn11p, Sps19p • Pyc1p, pyruvate carboxylase I with biotin attachment region :postranslational modification

  12. Results: Protein- Lipid InteractionsPhosphotidylinositides Structural component of membranes and as second-messengers regulate several cellular processes • Delivery: Liposomes consist of PC, biotin-DHPE and six different Ptd-Ins (5% w/w) • Detection: Streptavidin conjugated Cy3 • 103 known proteins: • 37 common targets (15 kinases) for all six Ptd-Ins • 8 to 34 protein targets specific for each Ptd-Ins • 61 membrane associated protein , 5 involved in lipid metabolism (Bpl1p), lipid modification (Kcs1p) or predicted membrane/lipid associated function • Lipid signalling in homeostasis Frm2p interacts with PI(3,4,5)P3

  13. Detection of protein-Ptd-Ins interactions on yeast proteome chips a-GST Probe PI(3)P PI(4,5)P2 PI(4)P PI(3,4)P2

  14. Selective binding of different Ptd-Ins to proteins Localization Function Target

  15. A B Rim15p Sps1p YGL059Wp Gcn2p Rim15p Hxk1p Eno2p BSA GST PI(4,5)P2 0.5mg 0.25mg 0.12mg 0.06mg 0.03mg 0.015mg PI(3)P PI(4)P PI(3,4)P2 PI(4,5)P2 PI(3,4,5)P3 PC C D Chip Membrane PI(3)P PI(3,4)P2 PI(4)P PI(4,5)P2 PI(3,4,5)P3 PC Rim15p Rim15p PI(3)P PI(4)P PI(3,4)P2 PI(4,5)P2 PI(3,4,5)P3 PC Rim15p Relative Intensity 0.5 mg 0.2 mg 0.05 mg 100 mm 5000 mm

  16. Data Analysis • Flag contaminated data points • Compare and scale signals from different experiments with respect to each other • Compute neighborhood subtracted signals • Create “hit list” • Look at differences between replicate samples both green (probe) and red (GST-protein amount) • Choose cut-off value for green signal [G=(G1+G2)/2] • Visual check for further input • Normalization • Compute ratios of green/red signal • Compute errors • Compute confidence limits for ratios

  17. Future Data Analysis • Visual and computer assisted signal detection-quantification, “hit list” generation • Search for common sequence motifs in “hits list” • Web interface to retrieve “hit lists” and associated image data

  18. Conclusions • A high-throughput protein purification, high-density protein microarraying and protein interaction detection protocol was developed • 1st entire eukaryotic proteome on chip • Protein-protein, protein-nucleic acid, protein-lipid, protein modifications, and small molecule –protein interactions can be screened • Unique approach to study biomolecule-protein interaction • Structural and functional categorization of yeast ORFs based on new findings

  19. Future Directions • Complete ORF clone collection • Improve chip fabrication, storage conditions, probe labeling and signal detection protocols • Screen proteome chip for other interactions • Enzymatic assays on proteome chips

  20. Collaborators • Gerstein Lab MB&B • Ronald Jansen • Ning Lan • Mark Gerstein • Kenneth Nelson • NCSU Fungal Genomics Laboratory

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