Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes

In
recent years, cross-linking mass spectrometry (XL-MS) has made
enormous strides as a technology for probing protein–protein
interactions (PPIs) and elucidating architectures of multisubunit
assemblies. To define conformational and interaction dynamics of protein
complexes under different physiological conditions, various quantitative
cross-linking mass spectrometry (QXL-MS) strategies based on stable
isotope labeling have been developed. These QXL-MS approaches have
effectively allowed comparative analysis of cross-links to determine
their relative abundance changes at global scales. Although successful,
it remains challenging to consistently obtain quantitative measurements
on low-abundant cross-links. Therefore, targeted QXL-MS is needed
to enable MS “Western” analysis of cross-links to enhance
sensitivity and reliability in quantitation. To this end, we have
established a robust parallel reaction monitoring (PRM)-based targeted
QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker
disuccinimidyl sulfoxide (DSSO), permitting label-free comparative
analysis of selected cross-links across multiple samples. In addition,
we have applied this methodology to study phosphorylation-dependent
conformational dynamics of the human 26S proteasome. The PRM-based
targeted QXL-MS analytical platform described here is applicable for
all sulfoxide-containing MS-cleavable cross-linkers and can be directly
adopted for comparative studies of protein–protein interactions
in various cellular contexts.