Session: Parallel session 9 - Top-down and Structural analysis

Pioneering Cross-linking Enhancing Approach: Increasing PhoX Efficiency to Unravel Protein-Protein Dynamics in Photosynthetic Membranes.

Introduction:

Membrane proteins are vital for cell thriving, and proper regulation of these systems is required in plants under variable light. Crosslinking (XL) mass spectrometry (MS) is a key tool for studying protein structures and interactions. Phosphate-affinity-based crosslinker (PhoX) allows easy post-crosslinking sample processing. However, PhoX efficiency is reduced for membrane proteins due to repulsion forces, thus being impermeable to enclosed systems. We present an XL enhancer which increase PhoX yields, boosting identifications even in whole cells.

Methods:

Crosslinking with PhoX was performed on photosynthetic membranes from Spinach (S. oleracea) and A. thaliana, and on cells from C. reinhardtii and P. tricornutum, whether in presence or not of the enhancer. Chlorophyll fluorescence measured the functional impact on photosynthetic membranes. Crosslinked proteins were identified by mass spectrometry and bottom-up proteomics, and fitted into PDB cryo-EM models. Interaction maps were generated using xiNET viewer.

Results:

XL peptide identifications increased with the enhancer, especially in cell samples. Spectral match counts and precursor intensity also rose. PhoX/protein ratio and sample's source affected the enhancing, lowering at high ratios. PhoX concentrations from 0.25 to 2 mM reduced photosynthetic membrane functionality, while the enhancer showed no effect below 0.1 M. Also, artifact crosslinks appeared at 2 mM PhoX due to distance incongruences in protein models. Cell crosslinking was not feasible unless the enhancer was applied.

Conclusion:

The enhancer increased crosslink identifications without requiring special protocols or damaging photosynthetic complexes. Using low-cost enhancers can extend PhoX yielding, mitigating its negative effects. This methodology shows potential for broader applications beyond photosynthesis, such as investigating protein interactome dynamics in organelles like mitochondria, and complex systems like viruses or cells.