Approaches for assessing and discovering protein interactions in cancer

Abstract

Significant insight into the function of proteins can be delineated by discovering and characterizing interacting proteins. There are numerous methods for the discovery of unknown associated protein networks, with purification of the bait (the protein of interest) followed by mass spectrometry as a common theme. In recent years, advances have permitted the purification of endogenous proteins and methods for scaling down starting material. As such, approaches for rapid, unbiased identification of protein interactomes are becoming a standard tool in the researchers toolbox, rather than a technique that is only available to specialists. This review will highlight some of the recent technical advances in proteomic-based discovery approaches, the pros and cons of various methods and some of the key findings in cancer-related systems.

Figure 1. Examples of novel, sensitive directed approaches for assessing protein-protein interactions.

A. FRET (Fluorescence Resonance Energy Transfer) involves conjugating fluorophores to the two proteins investigated. If the two proteins are in close proximal space (within 10nm), energy transfer occurs and emission can be measured. B. PLA (Proximity Ligation Assay) involves attaching oligonucleotides to a substrate, in this case antibodies. Two antibodies against two potentially interacting proteins are combined and if the two proteins are associated with each other, the oligunucleotides can be ligated and detected by PCR based methods. Both of these methods require prior knowledge of the putative interacting partner proteins.

Figure 2. Two methods for global identification of associated proteins.

A. The traditional tagging approach, where the protein of interest expressed as a fusion with a tag. Large-scale cell culture is employed and the tag is purified by immunoprecipitation. The precipitated proteins are resolved by gel electrophoresis and enriched protein bands specific to the experimental condition are excised and identified by MS. B. QUICK (quantitative immunoprecipitation combined with knockdown), involves SILAC labelling cells and transfecting either siRNA to the specific target or a control siRNA. Endogenous protein is pulled down and the two conditions are quantitative compared to identify proteins that are enriched and lost when the target is silenced.

Figure 3. RIME: Rapid Immunoprecipitation-MS of Endogenous protein.

RIME is a quick and endogenous approach for identifying associated proteins from limited starting material. A. The method is shown and involves cross-linking of complexes, followed by affinity purification using an antibody against the endogenous protein. The sample is split for ChIP-seq or for stringent washes, protease digestion and MS. B. Example of data from ER RIME. The data shows ER interactors identified in three independent replicates, but not present in any of five control experiments. The length of the line represents the MASCOT score and associated proteins are categorized based on molecular function. The data is reproduced with permission from: Mohammed et al, Cell Reports, 2013, 3: 342-349.