ac1c05298_si_004.xlsx (114.8 kB)
Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes
datasetposted on 2022-02-23, 09:04 authored by Clinton Yu, Xiaorong Wang, Lan Huang
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.
various cellular contextsstable isotope labelingrelative abundance changesmade enormous strideslinking mass spectrometryhuman 26s proteasomedifferent physiological conditionstargeted quantitative strategyfree comparative analysisprobe conformational dynamicsdependent conformational dynamicsvarious quantitative crosslinker disuccinimidyl sulfoxidebased targeted qxltargeted qxlinteraction dynamicsdefine conformationalstrategies basedcomparative studiesstudy phosphorylationselected crossremains challengingrecent yearsprotein complexesmultisubunit assemblieslinked peptidesglobal scalesenhance sensitivityelucidating architecturesdirectly adoptedcleavable crossalthough successfulabundant cross