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

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.

Affinity capture and MS analysis is a powerful method for the analysis of protein-protein complexes. A common method is TAP-tagging. This depends on the affinity isolation of a 'bait' protein containing a double tag. However, traditional TAP-tagging requires a strictly serial processing and this can compromise recovery, especially from low abundance or unstable complexes. Here we propose a modified affinity capture technique. We again use a 'bait' protein with two separate tags, but process cell extracts in parallel using columns for each tag. To discriminate between specific and non-specific binding we use stable isotope labelling with amino acids in cell culture (SILAC). We call this SILAC-iPAC. We will also explore the potential for using cleavable cross-linking in combination with SILAC-iPAC to isolate and characterize transient interactors. Our initial 'proof of principal' experiments will use DT40 cell-lines containing TAP-tagged protein engineered to express the tagged protein from the tag-modified endogenous gene by homologous recombination. An integral part of our proposal is also to develop an associated software package for the facile analysis of data generated by this method. We envisage that our method will be of wide applicability to many situations in cell biology and biochemistry where understanding and analysing protein-protein complexes is essential.

The main advance form this project will be the development of SILAC-iPAC and the production and dissemination of associated software. Full descriptions of this work will be published in peer reviewed litterature and presented at academic conferences. Biochemistry/cell biology community: Protein-protein interactions lie at the explanatory heart of most key biochemical and cell biological processes including, but not limited to: cell metabolism, DNA repair, gene expression and signal transduction. Developing accurate, sensitive and robust proteomic methods, including associated software for the analysis and characterisation of these complexes is a major priority. Databases that catalogue these structures are also essential. Proteomics community (both academic and industrial): There are already close collaborations between the University of Cambridge and several mass spectrometry vendors and proteomics software vendors including Thermo Finnigan, Applied Biosystems, Waters and Matrix Science. The proteomics community as a whole will benefit from the development of such widely applicable methodology and associated software as well as the generted databases. KSL has collaborated with Matrix Science and Applied Biosystems for many years including Applied Biosystems contribution to a BBSRC award which resulted in the first developments of organelel proteomics methods. We envisage that such collaborations with mass spectrometry vendors will continue throughout the period of this grant, and will result in us working with the appropriate vendors to incorporate them into the workflow. Pharmaceutical industry: The PI has given talks frequently to the pharmaceutical industry on cell biology and ion channel topics including Glaxo Smith Kline and Pfizer, and has numerous contacts with these industrial research groups. KSL has discussed proteomic methodology with Glaxo Smith Kline, Astra Zeneca and also Genentech. The methodologies developed within the context of this proposal are so fundamental that they could underpin many area of pharmaceutical research.