Yeast%20Proteome%20Chip

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Yeast Proteome Chip

• Global Analysis of Protein Activities Using
  Proteome Chips

Snyder Lab Zhu, Bilgin, Bangham, Hall,
Casamayor, Bertone, Bidlingmeier, Snyder
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Why Develop Protein Microarray-Chip Technology?
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Applications of Biochips

• DNA microarrays
• Gene expression analysis
• Genotyping
• Toxicogenomics
• Pharmacogenomics
• Diagnostics

• Protein microarrays
• Protein expression analysis
• Drug discovery
• Clinical diagnostics
• Others emerging

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Yeast Proteome Chip

• First Protein chocolate Chip

2.99
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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 gt1 copies
• To complete the collection
• 300 new unique clones sent for sequence
  confirmation
• 950 low quality sequencing

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Experimental Approach
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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

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• 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
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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

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Detection of different interactions on yeast
proteome chips
PI(3,4,5)P3
PC
Calmodulin
Genomic DNA
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Results Protein- Protein Interactions
Calmodulin

• Known interactions (4/8)
• Cmk1p, Cmk2p type I, type II calcium/calmodulin-d
  ependent 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

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Results Protein- Lipid InteractionsPhosphotidy
linositides

• 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

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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
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Selective binding of different Ptd-Ins to
proteins
Localization
Function
Target
PI(4,5)P2 RNA export Several RNA associated proteins
PI(3)P Vacuolar protein sorting Stp22
PI(4)P Plasma membrane Several membrane associated proteins
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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
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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(G1G2)/2
• Visual check for further input
• Normalization
• Compute ratios of green/red signal
• Compute errors
• Compute confidence limits for ratios

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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

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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

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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

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Collaborators

• Gerstein Lab MBB
• Ronald Jansen
• Ning Lan
• Mark Gerstein
• Kenneth Nelson
• NCSU Fungal Genomics Laboratory