PhoXplex: Combining Phospho-enrichable Cross-Linking with Isobaric Labeling for Quantitative Proteome-Wide Mapping of Protein Interfaces

Integrating cross-linking 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, the 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 cross-linkers
has greatly improved the detectability of protein interfaces in a
proteome-wide scale, facilitating global protein–protein interaction
mapping. Therefore, bringing together enrichable cross-linking 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 cross-linking
with TMT labeling 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 cross-links and long loop-links in total including potentially
novel interactions. Overlaying AlphaFold predictions and disorder
protein annotations enables exploration of the quantitative cross-linking
data set, 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.