Proteome-Wide Mapping of Protein Conformational Changes by Enzymatic Phospho-Probing

Mass spectrometry (MS) is increasingly being utilized to monitor protein structural changes using small sample amounts. These methods involve labeling the protein surface with probes and analyzing the probe-labeled peptides by MS. However, the low specificity of the probing and high complexity of samples make it challenging to detect protein conformational changes on a proteome scale with high sensitivity. In this study, we developed an MS-based structural analysis method that utilizes site-specific probing with phosphate groups, taking advantage of the substrate recognition provided by protein kinases. The phosphate groups serve as enrichment handles, enabling selective extraction and highly sensitive analysis of labeled sites. We found that differences in substrate protein structure were reflected in in vitro phospho-probing efficiency, and based on these differences, we successfully identified protein conformational changes upon RNA digestion.