An improved mass spectrometric method for the analysis of protein 'interactomes' using SILAC labeling and parallel affinity capture.

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Living cells contain a complex network of proteins that interact with each other - sometimes stably, sometimes more dynamically. Many of the key events that contribute to the maintenance and viability of the cell, and that are perturbed in disease depend on these interactions. Researchers have developed methods to identify and characterise these interactions. In general terms they often involve 'marking' or 'tagging' one of the proteins to be examined in such a way that the 'tagged' protein can be selectively isolated and the associated partners identified by a technique called mass spectrometry. However, current methods for this analysis are not always satisfactory and can be probematical when there are low levels of such proteins or when the interacting complexes are not stable. Here we propose a method we call SILAC-iPAC that combines several techniques, each of which has been used in other contexts, but never before in combination, that we believe will offer a significant improvement in the ability to identify protein-protein partners under these conditions. In addition, we propose to include specifically developed software to enable the easy and accurate analysis of any data obtained with the technique.