title: Developing new top-down mass spectrometry strategies for studying transient protein-protein interactions
Creators: name: Mateos Jimenez, Maria
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Mass Spectrometry (MS)
subject: Protein-Protein Interactions (PPI)
subject: Transient
subject: TPR2A:AcMEEVD
subject: Top-Down Strategy

description: Protein-protein interactions (PPI) have attracted much attention as potential drug targets since the publication of the Human Genome Report. Understanding normal protein function and its modulation via interaction with other molecules, including other proteins, is paramount to understanding human health and disease. Despite mass spectrometry (MS) typically providing lower resolution structural data than well-established techniques, it presents as a versatile alternative for the study of the more challenging targets such as transient PPI, which typically establish weak contacts and have a rather dynamic character. In this work, different top-down MS strategies were explored for the characterisation of a large transient PPI using a domain:peptide interaction as a proxy. More specifically, the TPR2A:AcMEEVD interaction was studied as a proxy for the transient PPI involving the co-chaperone HSP70-HSP90 organising protein (HOP) and the chaperone heat shock protein 90 (HSP90). The HOP:HSP90 interaction has previously been shown to support the survival of cancer cells, qualifying as an attractive cancer drug target. Firstly, alanine scanning was combined with native MS and ion mobility MS to evaluate the ability of this strategy to study the contribution of individual interface hot spots to the binding strength and gas-phase stability of the TPR2A:AcMEEVD complex. Kd estimations derived from MS measurements from wild-type and alanine mutants showed that every individual hot spot seems to be indispensable for the efficient binding of AcMEEVD to TPR2A. Collision cross-sections obtained from native ion mobility MS at low collision energy indicated that the decrease in binding strength was likely caused by loss of key electrostatic contacts rather than structural destabilisation, as major ion conformational changes were not observed. Gas-phase stabilities demonstrated a consistent modest stabilisation of the compact structure of the different variants, including wild-type, when they were bound to the peptide. Secondly, the supercharging method and ion pre-activation approach were applied to elucidate the location of the domain:peptide binding site by native top-down tandem electron capture dissociation (ECD). Ion mobility MS analysis suggested that adding 1.5 % m-nitrobenzyl alcohol to protein samples did not seem to disrupt the native-like confirmation of ion. Lower ECD fragmentation was observed in the inner α-helices H3 and H4 when TPR2A was bound to AcMEEVD, suggesting a protective role of the peptide. 36 peptide-bound protein fragment ions indicated the ability of the peptide to interact with both N and C terminal domains of TPR2A. Unfortunately, the orientation of the linear peptide within the domain:peptide interface could not be determined with the current workflow due to the current ambiguity associated with assignment of peptide fragment:protein fragment ions. Thirdly, a new strategy combining carbene protein footprinting with top-down tandem MS was developed to evaluate its ability to study transient PPI by mapping protein exposed surface from a top-down approach. The workflow for sodium 4-(3-Trifluoromethyl)-3H-diazirin-3-yl)benzoate (NaTDB) photoactivation was extended by using a LED light source (365 nm, 20 s) instead of a laser. The comparative experiment between TPR2A and TPR2A:AcMEEVD successfully located the binding site of TPR2A between residues 22 to 112, in accordance with literature. Finally, this strategy was further combined with the isotope depletion method, which allowed the detection of 41 ions more with respect to isotopically normal samples.