yeast proteome chip
Download
Skip this Video
Download Presentation
Yeast Proteome Chip

Loading in 2 Seconds...

play fullscreen
1 / 20

Yeast Proteome Chip - PowerPoint PPT Presentation


  • 178 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Yeast Proteome Chip' - didier


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
yeast proteome chip

Yeast Proteome Chip

Global Analysis of Protein Activities Using Proteome Chips

Snyder Lab

Zhu, Bilgin, Bangham, Hall, Casamayor, Bertone, Bidlingmeier,

Snyder

applications of biochips
DNA microarrays

Gene expression analysis

Genotyping

Toxicogenomics

Pharmacogenomics

Diagnostics

Protein microarrays

Protein expression analysis

Drug discovery

Clinical diagnostics

Others emerging

Applications of Biochips
yeast proteome chip1
Yeast Proteome Chip
  • First Protein “chocolate” Chip

$ 2.99

yeast proteome chip building up the yeast orf collection
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
chip fabrication probing and detection technical issues
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

slide8
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

analysis of the yeast proteome chip
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
slide10

Detection of different interactions

on yeast proteome chips

PI(3,4,5)P3

PC

Calmodulin

Genomic DNA

results protein protein interactions calmodulin
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
results protein lipid interactions phosphotidylinositides
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
slide13

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

slide14

Selective binding of different Ptd-Ins

to proteins

Localization

Function

Target

slide15

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

data analysis
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
future data analysis
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
conclusions
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
future directions
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
collaborators
Collaborators
  • Gerstein Lab MB&B
    • Ronald Jansen
    • Ning Lan
    • Mark Gerstein
    • Kenneth Nelson
    • NCSU Fungal Genomics Laboratory
ad