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

Abstract

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.

Figure 1.. General workflow for cross-link identification and targeted cross-link quantitation.

(A) Cross-links are identified by LC MSⁿ, and the resulting cross-link spectral matches are used to develop a target library and transition list for PRM analysis. (B) Cross-links are analyzed by targeted PRM analysis using CID to produce cross-link fragments while minimizing backbone fragmentation, simplifying the quantitation process. Transitions for each cross-link target are measured in Skyline.

Figure 2.. Workflow for investigating calyculin-dependent conformational dynamics of the 26S proteasome.

26S proteasomes were purified from control and calyculin-treated 293^HTBH-Rpn1 cells and cross-linked. Digested cross-linked peptides were then analyzed by LC MS/MS, LC MSⁿ, and PRM to determine proteasomal subunit abundances, phosphorylation, and cross-link identification and quantitation.

Figure 3.. Quantitation of purified 26S proteasomes.

(A) Relative abundance ratios (treated/control) of proteasome subunits as determined by LFQ measurements in MaxQuant. All subunit abundances normalized to Rpn1. Pair-wise correlation plots between (B) control replicates and (C) calyculin-treated replicates indicating good agreement between replicate experiments.

Figure 4.. XL-MS data of the 26S proteasome.

(A) 2-D cross-link map generated from CX-Circos (http://cx-circos.net) depicting interconnectivity of proteasome subunits within respective subcomplexes. (B) Mapping of cross-links to high-resolution 26S proteasome structure (PDB 5GJR). Cross-links with mapped Cα-Cα distances below 30 Å shown in green, and above 30 Å shown in red. (C) Histogram showing distribution of mapped cross-links by distance.

Figure 5.. PRM quantitation of the proteasome cross-link targets.

PRM quantitation in Skyline for cross-link targets corresponding to (A) Rpn2:K574-Rpn8:K28 and (B) Rpn1:K39-Rpt6:K330. Four transition ions (α_A²⁺, α_T²⁺, β_A²⁺, and β_T²⁺) for each cross-link target were measured to calculate relative cross-link abundances. Pair-wise correlation plots for K-K linkage abundances between (C) control replicates and (D) calyculin-treated replicates. (E) Distribution of cross-link abundance ratios in calyculin-treated versus control proteasomes.

Figure 6.. Analysis of statistically changed cross-links and mapping to the proteasome structure.

(A) Volcano plot depicting the distribution of changed (blue) and unchanged (orange) cross-links in treated versus control proteasome purifications. Cross-links that did not meet the p-value threshold of 0.10 are shown in gray. (B, C) Mapping of calyculin-dependent cross-links to high-resolution 26S proteasome structure (PDB 5GJR). Cross-links reduced in calyculin-treated proteasomes shown in red, cross-linked increased shown in green. 19S base assembly depicted in teal, 19S lid in orange, and 20S in purple.