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.

近年来，作为探究蛋白质-蛋白质相互作用（PPIs）和阐明多亚基组装结构的技术的交叉链接质谱（XL-MS）取得了显著的进展。为了定义蛋白质复合物在不同生理条件下的构象和相互作用动力学，基于稳定同位素标记的各种定量交叉链接质谱（QXL-MS）策略已被开发。这些QXL-MS方法有效地实现了对交叉链接的比较分析，以确定其在全局尺度上的相对丰度变化。尽管取得了成功，但持续获得低丰度交叉链接的定量测量仍然具有挑战性。因此，需要针对性的QXL-MS来实现质谱“Western”分析，以提高定量分析的灵敏度和可靠性。为此，我们建立了一个基于并行反应监测（PRM）的靶向QXL-MS平台，该平台使用含有亚磺酰基的质谱可切割交联剂二硫代琥珀酰亚胺（DSSO），允许对多个样本中选定的交叉链接进行无标记的比较分析。此外，我们还应用了这种方法来研究人类26S蛋白酶的磷酸化依赖性构象动力学。本文所述的基于PRM的靶向QXL-MS分析平台适用于所有含有亚磺酰基的质谱可切割交联剂，并可直接用于比较研究各种细胞环境中的蛋白质-蛋白质相互作用。