TAP(Tandem Affinity Purification) Billy Baader Genetics 677.

TAP(Tandem Affinity Purification)

Billy Baader

Genetics 677

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Protein-protein interactions

Protein Identification

20,000+ genes in humans

Millions of proteins

Understanding human biological processes



Protein IdentificationOther available methods-2D gel analysis-Labeling methods-Antibodies-Peptide tagging-Mass Spectrometry

What are some of the problems with these methods?









Problems with Classical Methods

Requires large amounts of protein

Limitations in the number of testable samples

Purification

Contamination

Time

How TAP works







TAP compared to other methods

Flag Tag– Small peptide tag

Natural protein levels vs. overexpressed proteins

Yeast Two Hybrid– Low level of overlap– Assays protein interactions



Protein internal structure

• Desire to understand molecular mechanismsSome proteins lack obvious enzymatic activity

Once proteins are discovered we would like to know how they work and interact

Possibilities-Crystillization-Electron Microscopy-Two Hybrid Assay-Chemical Cross-Linking



Functional organization of the yeast proteome by systematic analysis of protein complexes

Gavin et al.

Practical application of TAP and mass spectrometry on S. cerevisiae

Emphasizes the potential for a massive amount of information to be obtained through TAP

Potential of protein knowledge

“Whenever it has been possible to retrieve and analyze particular cellular protein complexes under physiological conditions, the insight gained from the analysis has been fundamental for the biological understanding of their function”

i.e. spliceosome, cyclosome, proteasome

Examined 25% of ORFs in yeastQuickTime™ and a

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Method for purificationTAP1. High affinity purification2. Elution3. Second affinity purification

Separate with gel electrophoresis

Digest with trypsin

Analyze with mass spectrometry





TAP tags

TAP cassette created through PCR

Insertion at the C-terminus of a selected yeast ORF by homologous recombination

Examined 1,739 genes





Homologous Recombination of TAP tag

Proteins purified from different organelles



Efficiency



Examining the data

Technical Bias against proteins below 15kDa

Possiblity of using different entry points to purify protein complexes

Comparison to literature

70% reproducability

Polyadenylation machinery

• Responsible for eukaryotic mRNA cleavage and polyadenylation

• Single entry point used: Pta1

• 12 of 13 known interactors and 7 new components





Reproducibility using various entry points

Polyadenylation machinery



Protein complex networks

Utilized an algorithm to automatically generate map

Links are between complexes sharing at least one protein





Protein Network

Orthologs

Examined the hypothesis that orthologous gene products are responsible for essential cellular activities

Orthologous complexes interact preferentially with other orthologous complexes

Nonorthologous complexes do not interact at as well with the orthologous complexes

The same relation is present between essential and non-essential complexes

Human/Yeast Orthologs

• Arp2/3– Cytoskeleton-associated complex

• Ccr4-Not– Involved in control of gene expression

• TRAPP– Transport protein particle– associated with Golgi body



Ortholog Comparison Results

Results

• Huge increase in number of proteome components

• TAP was responsible for a efficient identificaiton of low-abundance proteins as well as large complexes

• Differences in the aspects of protein interaction detected through TAP compared with Y2H

• Orthologous complexes appear to represent the building blocks of a ‘core proteome’



Advantages of TAP

• Simplicity

• Cleaner, more intact complexes

• Higher yield

• Low false negative rates

• Tags show little protein alteration



TAP vs. Y2H

• Analyzes complexes and can create protein network maps

• Analyzes more of the proteome

• Works in membrane proteins

• Works in many organisms

• Analyzes binary interaction between proteins

• Works with transient protein interactions

• Performed in vivo

These methods yield different information and should be used complementarily

PossibilitiesGavin et al. believe that there methods are one of the most efficient

and unambiguous routes towards the assignment of gene identity and function

Easy to analyze large amounts of protein complexes and assess there relation to each other

Increase in understanding of biological systems and their processesDrug discovery and usage may be greatly enhanced through this

knowledgeIdentification of a vast number of proteins and protein complexesOther techniques may be used to understand the function of these

proteinsA more complete understanding of the proteomes can hopefully be

developed

Questions

The review, when talking about TAP, said that it has been used to purify membrane bound protein complexes. I don't know a lot about protein purification, but I have always heard that purifying membrane proteins is notoriously difficult. Can you explain what make TAP better suited for this than other methods?

Questions

In the Review paper table 1 compares Flag and TAP; why is the fraction of successful purification (both with and without interacting proteins) higher for Flag? The other statistics in the table seem to make Flag a poor alternative, however, the fraction of successful purifications would seem to be an important percentage to raise. What is being done to improve this?

Questions



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TAP(Tandem Affinity Purification) Billy Baader Genetics 677.

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