Talk Title: Probing biopharmaceutical proteins and protein assemblies by hybrid mass spectrometry approaches
MAlbert J.R. Heck (Utrecht University) is scientific director of the Netherlands Proteomics Centre. A large emphasis of Heck’s group is on the development and applications of advanced mass spectrometry based proteomics technologies. Heck introduced TiO2 and Ti4+-IMAC based technologies for phospho-enrichment. Heck pioneered the use of alternative proteases and hybrid peptide fragmentation techniques (e.g. EThcD). Heck also introduced 15N labeling in multicellular organisms and the cost-effective dimethyl labeling. Heck’s proteomics research focuses for a large part on cancer, stem cells and immunology. Besides the proteomics efforts, the group of Heck is also known for its expertise in mass spectrometry based structural biology, using native mass spectrometry, cross-linking and/or HD exchange mass spectrometry. The Heck-lab developed dedicated instruments for the analysis of intact proteins and protein complexes, with most recently a new high-mass Orbitrap, a serious breakthrough for top-down proteomics and native mass spectrometry.
Heck is recipient of awards such as the HUPO Discovery Award (2013), and the Proteomics Pioneer Award from the European Proteomics Association (EuPA, 2014). In 2016 he received the ACS Frank H. Field and Joe L. Franklin Award. In 2014 he became elected member of EMBO and the Royal Netherlands Academy of Sciences and Arts (KNAW).
Abstract: Probing biopharmaceutical proteins and protein assemblies by hybrid mass spectrometry approaches
Albert J. R. Heck 1,2
1 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
2 Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Mass Spectrometry based proteomics has played a pivotal role in revealing the plethora of protein interactions and post-translational modifications that take place inside a cell, wherein proteins form protein assemblies and/or signalling networks. Especially using affinity purification of a tagged proteins followed by mass spectrometric analysis of its binding partners a wealth of data has been gathered revealing the all-embracing protein networks present in cells. Following the charting of all these interactions, a next step will be to now gather more in-depth structural and functional information on these individual protein assemblies. This may come from in-depth high-resolution structural models, as well as detailed information on how they function and dynamically evolve during cellular perturbations. Mass spectrometry may also contribute to this next level of protein interaction analysis although it does require partly different and novel approaches. To contribute to this emerging new area in proteomics, our group is developing new methods using native mass spectrometry and cross-linking mass spectrometry with the aim to bridge the gap between interaction proteomics and structural biology. These new innovations and applications of them in interaction proteomics will be central in this presentation.
In the first part of the talk native mass spectrometry and its applications in probing
protein assemblies and interactions will be described, focusing on examples wherein the dynamic assembly of a protein complex involved in the circadian timing in cyanobacteria will be highlighted. Herein, by using a combination of native, HD exchange and cross-linking mass spectrometry and cryoEM, we were able to define a novel structural model improving our understanding of the circadian rhythm. Additionally, novel developments in MS instrumentation for native MS will be highlighted, especially a new Orbitrap based instrument that offers high-sensitivity and mass resolution, allowing an in-depth detailed analysis of glycoproteins, viruses and even whole intact ribosomes.
The second part of the talk will highlight our recent work on cross-linking mass spectrometry. Cross-linking combined with mass spectrometry (XL-MS) provides another powerful approach to probe the structure and interaction profile of protein assemblies. Up to now XL-MS has been primarily limited to the characterization of purified protein assemblies. We have set out to develop XL-MS methods aimed at probing protein interactions at the proteome level, using complete cell lysates or whole organelles as starting material. We, therefore, combined several novel innovative methods to address some of the hurdles in this field. These innovation include the use of a low energy CID cleavable cross-linker, novel hybrid pepetide fragmentation and acquisition strategies and a dedicated software suite, termed XlinkX. We applied this novel XL-MS strategy to lysates of E. coli lysate and human HeLa cell lines, and to mammalian mitochondria and nuclei. In each of these studies we successfully identified thousands of cross-links. Many of the identified cross-links could be validated by mapping them on available high-resolution structures, but the data also provide information on assemblies for which no high-resolution structures are available, and even reveal new protein interaction networks.