Probing protein assemblies and interactions by hybrid mass spectrometry approaches
Authors: Heck Albert J.R. 
Published: November 2023

Mass Spectrometry based proteomics has played a pivotal role in revealing the plethora of protein interactions 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.Support or Funding InformationThis work was supported by The Netherlands Organisation for Scientific Research (NWO) via the Roadmap Initiative Proteins@Work (project number 184.032.201), and through the European Union Horizon 2020 programme FET‐OPEN project MSmed, project number 686547.