The protein–protein interaction network of the eukaryotic nucleus
Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
In proteomics, mass spectrometry has traditionally been used to profile the identity, abundance and modification state of proteins within complex biological samples, providing a systems-level means to examine cellular function. Until very recently, interrogation of protein-protein interactions on a global scale has not been technically possible, with the tools of choice being low-throughput and high-noise techniques such as affinity-based pulldowns (AP-MS) and proximity labelling (such as BIO-ID). Cross-linking mass spectrometry (XL-MS) couples the use of novel cross linkers such as DSSO (membrane permeable, amino acid reactive, covalent bond forming, mass-spec cleavable) with high mass accuracy/resolution tandem mass-spectrometry. XL-MS can generate high-throughput, unbiased and rich protein interaction networks from complex samples. It provides information beyond just identifying protein interactors or complex components, but also structural information at the protein and complex level. However, the dynamic range of protein expression in the eukaryotic cells is very large - functionally important yet low abundance proteins, such as those found in the nucleus, are usually underrepresented in proteomic and hence XL-MS studies. This project aims to generate the first high coverage and high confidence interaction network of a eukaryotic nucleus, to provide fundamental insights into the architecture and dynamics of nuclear processes. Nuclei from actively dividing yeast were isolated by enzymatic cell wall digestion, mechanical cell disruption and sucrose density gradient centrifugation. SILAC-based quantification was used to calculate enrichment and purity of nuclear proteins relative to a heavy labelled whole cell lysate. Nuclei were highly enriched and showed significant depletion of common cytosolic and other organellar proteins. XL-MS data will be presented to show a draft of the eukaryotic nuclear protein-protein interaction network.