'Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry'

The main aim of our research in to develop and apply mass spectrometry based methods to investigate structure-function relationship of individual proteins up to the complete assembly of proteins in their cellular context.

In the first part of the talk I will describe a novel integrated workflow that allows us to identify thousand of cross-links from a variety of endogenous protein complexes in human cellular lysates. Our approach is based on the application of MS-cleavable cross-linkers, hybrid peptide fragmentation acquisitions (e.g. CID and ETD), and a novel dedicated search engine XlinkX that allows efficient cross-link identification against a complete human proteome database. This approach allowed us to detect 2179 unique cross-links in HeLa cell lysates, representing one of the largest cellular cross-linking MS dataset up to date. The confidence of our cross-linking results is validated by using a target-decoy strategy and through mapping the observed cross-link distances onto existing high-resolution structures. Intriguingly, our data revealed new structural information on many protein assemblies, capturing also dynamic interactions for instance the ribosome in contact with different elongation factors.

In the second part of the talk I will describe how we combine native mass spectrometry, ion mobility mass spectrometry, bottom-up proteomics and cross-linking mass spectrometry to study the interplay between two kinases and their joint scaffold; i.e. Aurora A, Plk1 and Bora, which are essential for mitotic entry after DNA damage-induced cell cycle arrest. Plk1, Aur-A, and Bora are known to engage in mutual phosphorylations and stable interactions but the linkage between these events is not fully understood. We studied their interdependence in vitro by integrating an array of complementary mass spectrometric techniques. We show that the N-terminus of Bora (BoraNT) efficiently triggers Plk1 activation and immediately forms a stable complex with Aur-A, suggesting that the complex is the Plk1-activating entity. Activated Plk1 and Aur-A subsequently phosphorylate BoraNT up to 22 times. This hyperphosphorylation, which appears to be accompanied by a structural rearrangement of BoraNT, proceeds via a well-ordered sequence of site-specific modifications and is the precondition for stable Plk1/BoraNT complex formation. Collectively, we provide a first molecular description of the interdependent transient and stable interactions governing the Aur-A/Bora/Plk1 interplay.