PhoXplex: Combining phospho-enrichable crosslinking with isobaric labelling for quantitative proteome-wide mapping of protein interfaces

ABSTRACT: Integrating crosslinking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable crosslinkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable crosslinking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable crosslinking with TMT labelling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 crosslinking events including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative crosslinking dataset, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Graeme Benstead-Hume  

LAB HEAD: Jytoi Choudhary

PROVIDER: PXD052480 | Pride | 2024-10-21

REPOSITORIES: Pride