Yeast proteome chip
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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 . Why Develop Protein Microarray-Chip Technology?. DNA microarrays Gene expression analysis Genotyping Toxicogenomics

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Yeast Proteome Chip

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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


Yeast proteome chip

Why Develop Protein Microarray-Chip Technology?


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


Experimental approach

Experimental Approach


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


Yeast proteome chip

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


  • Yeast proteome chip

    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


    Yeast proteome chip

    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


    Yeast proteome chip

    Selective binding of different Ptd-Ins

    to proteins

    Localization

    Function

    Target


    Yeast proteome chip

    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


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