General Technique
1) Protein-based affinity isolation of complexes. Epitope tags (short stretches of amino acids fused to a protein of interest) provide a very effective way to rapidly isolate a protein of interest along with its associated proteins from crude cell extracts. However, to preserve protein interactions, relatively mild isolation conditions must be used, and this can result in a high level of contaminant proteins in the final sample. Two strategies are used to circumvent this problem. The first approach is to modify a target protein with two epitope tags in tandem that can be used to purify the protein complex in two successive steps. This approach often yields highly purified protein complexes that can be analyzed directly by mass spectrometry. One commonly used tandem tag consists of an IgG binding domain (ProtA) separated from a calmodulin binding domain (CBD) by a TEV protease recognition site. To isolate protein complexes, extracts are incubated with IgG Sepharose beads to allow the ProtA tag to bind to IgG. After washing, the bound proteins are eluted by incubation with TEV protease which cleaves the protein at the TEV recognition site. Eluted protein is then bound to a calmodulin Sepharose column via the CBD epitope. Binding to calmodulin is dependent on the presence of calcium. After washing the purified protein complex, it is eluted by addition of a calcium chelator such as EGTA.
The second approach involves using quantitative mass spectrometry to analyze the composition of a partially purified protein complex and a relevant control purification in which the complex of interest is not enriched. For example IgG Sepharose can be used to purify proteins from an extract containing a ProtA tagged protein and from an extract containing the wild type (untagged) version of the protein. Proteins in the two samples are differentially labeled with stable isotopes and analyzed by mass spectrometry (see quantitative proteomics). In this approach, specifically interacting proteins can be distinguished from nonspecifically co-purifying proteins by their abundance ratios. In addition, because complexes can be analyzed after a single step purification, the potential to detect weakly associated proteins is enhanced. This approach also has been used to characterize the composition of organelles.
2) DNA affinity isolation of transcription complexes. DNA affinity chromatography is a powerful way to isolate transcriptional regulatory complexes. However, complexes isolated by single-step DNA affinity chromatography have a high amount of nonspecifically co-purifying proteins. To circumvent this problem we have used stable isotope tagging and mass spectrometry to quantitatively compare the composition of partially purified DNA binding complexes, and control purifications in which the complex of interest is not enriched. This approach is analogous to that described above for the analysis of protein complexes. For example, a DNA binding transcriptional regulatory complex can be isolated from cell extracts by incubating cell extracts with immobilize DNA binding sites. For comparison, the same extract is incubated with immobilized binding sites containing mutations that abolish binding or transcription activity. Proteins in the two samples are differentially labeled with stable isotopes and analyzed by mass spectrometry as described above.
Purpose/use/application of the technique:
Defining the components of protein complexes and their interactions is an essential component of understanding biological processes. Affinity purification of protein complexes followed by mass spectrometric analysis provides an efficient and effective way to characterize these interactions. Furthermore, iterative use of the approach allows us to decipher networks of protein interactions.
Example(s) of projects at ISB that use this technique:
Transcriptional regulatory complexes—Ranish
Protein phosphatase networks — Gingras
Halobacterium transcription factor complexes — Baliga
Peroxisomes — Aitchison, Marelli
Nups — Aitchison, Dilworth,
Karopherins — Leslie
To comprehensively characterize protein interactions networks by the affinity purification/mass spectrometry approach, it essential that complexes remain intact during the isolation procedure. Currently, we are testing new approaches for the rapid isolation of complexes from cells. These include new epitope tags that allow efficient isolation of intact protein complexes, and the use of in vivo cross linking agents, as a way to preserve interactions during purification. In addition we are improving quantitative mass spectrometry approaches for the study of protein complexes by applying new isotope labeling strategies. Representative publication(s):
(Ranish et al., 2004; Ranish et al., 2003)
(Flory et al., 2004; Marelli et al., 2004)
(Brand et al., 2004; Himeda et al., 2004)
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