Enhancing Inter-link Coverage in Cross-Linking Mass Spectrometry through Context-Sensitive Subgrouping and Decoy Fusion

Abstract

In cross-linking mass spectrometry, sensitivity and specificity in assigning mass spectra to cross-links between different proteins (inter-links) remains challenging. Here, we report on limitations of commonly used concatenated target-decoy searches and propose a target-decoy competition strategy on a fused database as a solution. Further, we capitalize on context-divergent error rates by implementing a novel context-sensitive subgrouping strategy. This approach increases inter-link coverage by ~ 30 - 75 % across XL-MS datasets, maintains low error rates, and preserves structural accuracy.

Structural Host-Virus Interactome Profiling of Intact Infected Cells

“…SHVIP integrates well with AF2 to inform structural predictions at interactome scale [19][20][21] delivering residue-residue connections as corroborative evidence to purely in silico analyses. Enhancing interactome-wide AF2 modeling with SHVIP is particularly valuable from a virological point of view because (1) AF2-only predictions of dimers involving viral proteins have generally lower quality than those of host proteins 59 (Figure 4) and (2) experimental structures of host-virus protein complexes are scarce for HSV-1 and many other viruses.…”

“…These XL-MS data reveal PPIs and yield insights into in situ protein complex structures and binding interfaces. For example, they have successfully been used to validate [19][20][21] and augment 22 structural models obtained through AlphaFold2 (AF2) 23 . Importantly, XL-MS is applicable to endogenous proteins in their native cellular environment and can provide PPI networks of intact organelles 24,25 , cells 20,26 , and viral particles 27 .…”

“…For example, they have successfully been used to validate 19–21 and augment 22 structural models obtained through AlphaFold2 (AF2) 23 . Importantly, XL-MS is applicable to endogenous proteins in their native cellular environment and can provide PPI networks of intact organelles 24, 25 , cells 20, 26 , and viral particles 27 . However, previous XL-MS studies on pathogen-host interactions from intact infected cells have been limited in sensitivity and identified fewer than 50 PPIs 28 likely due to the complexity and abundance of the background host proteome.…”