Utilizing Platinum(II)-Based Cross-Linker and Two-Stage Data Analysis Strategy to Investigate the Allosteric in Glycogen Phosphorylase

Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein–protein interactions (PPIs). Throughout the entire workflow, the utilization of cross-linkers and the interpretation of cross-linking data are the core steps. Cisplatin, as a well-known anticancer drug, has been previously demonstrated for its capability and advantages as a cross-linker. However, the complexity of platinum(II) cross-linked products and the lack of suitable software for data interpretation have hindered its further application. In this work, a two-stage data analysis strategy for platinum(II) cross-linked peptides has been developed and demonstrated on a pair of phosphorylation-induced allosteric systems, glycogen phosphorylase (GP) b and a. This two-stage data analysis strategy takes into account the identification of various types of Pt(II)-containing fragment ions and incorporates the unique isotope distribution properties of Pt(II)-based cross-linkers to eliminate false-positive data and achieve accurate identification of Pt(II)-based cross-linked peptides. The Pt(II)-based cross-linking results allow the capture of structural differences between GPb and GPa at the N-terminus and the tower–tower helices interface, which is consistent with the X-ray crystallography structure as well as our previous HDX-MS results. In addition, it also complements the structure of noncrystallizable regions. Finally, through discussion of existing data search engines and issues in spectral analysis of Pt(II)-based cross-linked peptides, we put forward proposals for follow-up software design, cross-linker developments, and guidance for the application of platinum(II)-based drugs. Overall, Pt(II)-based XL-MS can be a useful tool to complement both experimental and computational structural biology.