Download Proteomics

Protein-Protein Interactions
• High-throughput strategy
– Prediction from sequence
• In silico analysis
– Protein A from species A: domain 1 and 2
– Protein 1’ and protein 2’ from species B
• Recognition  sequence homology
– Yeast two-hybrid screen of whole genome
– Tagged protein
• Tandem affinity purification (TAP) + MS
• Immunoprecipitation + MS
– Ab to target proteins
– Pooling assay
• Biochemical functional assay
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http://depts.washington.edu./sfields/
Y2H: basic design
•
If the two proteins interact, the reporter gene (here: HIS3) is
switched on and the diploids can grow on -His plates:
•
If the two proteins don't interact, the reporter gene remains
inactive and the cells can't grow on -His plates:
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Discovery Q
• Y2H protein interaction inside nucleus
of yeast cell. Is it OK?
• What is the proper control?
• Is it restricted to yeast proteins only?
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Large-scale Y2H
• Yeast genome
– Array screening
• Much more time- and labor- intensive
• More positive identification
– Library screening
• Reasonable time and effort
– Bioinfomatics platform
• http://portal.curagen.com
Uetz, et al., Nature 2000 403, 623-627.
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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DIP search
• Database of interacting proteins
Start/root node
1st shell nodes
2nd shell nodes
Interactions
Color: reliability
Width: no. of exp.
http://dip.doe-mbi.ucla.edu/
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Protein network
• Built by association
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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Interaction between groups
• Crosstalk between and within functional groups
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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By location
• Grouped by cellular compartments
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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Prediction of functions
• Guilty-by-association
Ypt1
Akr2
Yip1
YPL246C
YHR105W
YGL161C
Vam7
Endocytosis
Pep12
Vesicle transport &
membrane fusion
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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Tag-protein + MS
• Co-precipitation
– Tandem-affinity
purification
– SDS-PAGE
• Mass spectrometry
• Bioinformatics
Kumar and Snyder, 2002 Nature 415, 123-124
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Pooling functional assay
• Biochemical assay for activity
– 6144 GST-ORF strains
– 64 pools of 96 fusions/plate each
– Pools of 12 columns and 8 columns
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12
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8
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Priori and Potentials
• Priori
– The GST-ORF is functional
– Soluble after extraction
– Remain functional
• Retains other required components when purified
• Fast and sensitive
• Potentials
– Determine the range of the substrate proteins
– Identifying gene leads to the binding of
particular molecule, ligand, or drug.
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Next Week
• Please read Text p. 183-187.
– 1:30 pm at room A105
– Advanced handout?
• Paper discussion
– Whatever we did not finish today
• Homework assignment
– How far did you get?
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