Oral Presentation 24th Annual Lorne Proteomics Symposium 2019

Large scale crosslinking mass spectrometry to map the yeast nuclear interactome (#3)

Tara K Bartolec 1 , Daniela-Lee Smith 1 , Marc R Wilkins 1
  1. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Randwick, NSW, Australia

Proteome scale crosslinking mass spectrometry (XL-MS) couples the use of novel cross linkers with specialised mass-spectrometry (MS) methods to generate high-throughput and rich protein-protein interaction networks from complex samples. Application of XL-MS to the quantitative study of dynamic interactomes (for example across time, or between wild-type and gene knockout systems) will reveal new insights into cellular signalling and function. However, the large dynamic range of protein expression levels within eukaryotic cells presents a challenge in traditional proteomic, and furthermore XL-MS, studies; low abundance yet functionally important nuclear proteins are generally underrepresented. Here we will present the first large-scale yeast nuclear XL-MS protein interactome. Nuclei from actively dividing wild-type yeast were isolated by enzymatic cell wall digestion, mechanical disruption and sucrose density gradient centrifugation. Intact nuclei were then crosslinked using the cleavable crosslinker DSSO. Offline strong cation exchange gradients were optimised for crosslinked peptide enrichment and separation, alongside complementary online reverse phase liquid chromatography optimisation. A highly interconnected protein interactome was generated containing 2,091 crosslinks (identified at a 5% FDR) and representing 1,148 proteins. 1,625 crosslinks involved an interaction where at least one partner was annotated as nuclear, and 922 with both interactors as nuclear (representing 823 and 431 proteins, respectively). Over one third of all crosslinks could be matched to a known BIOGRID physical interaction, and clusters of known complexes including the RNA polymerase, chromatin structure remodelling (RSC) and preribosome complexes emerged within the network. This high depth dataset represents a significant foundation towards the broader goal of proteome-scale, quantitative XL-MS studies of the dynamic yeast nuclear interactome.