Figure 1. LiP–MS detects protein–protein interactions in purified systems.

(A) Schematic of LiP–MS workflow. Proteins are extracted from an experimental model, such as tissues, human cells, bacteria, yeast, viruses, or biofluids, under native-like conditions. The extract is then exposed to a protein of interest (treated) or not exposed (control) and subjected to limited proteolysis with proteinase K. Under LiP conditions, proteinase K cleaves solvent-exposed, accessible, and flexible regions, thus generating protein fragments that may differ between the treated and control samples for an interactor of the spiked-in protein. These protein fragments are digested by trypsin under denaturing conditions to produce peptides that are measurable by bottom-up proteomics. By comparing differential peptides between the treated and control samples, interactors of the protein of interest can be identified. (B) Structures of preRSVF (left, PDB: 4JHW) (McLellan et al, 2013) and postRSVF (right, PDB: 3RRR) (McLellan et al, 2011). Known antigenic sites are shown both on the protein structure and in isolation (middle). Blue indicates antigenic site Ø, targeted by antibodies D25 and 5C4. Red indicates antigenic site II, targeted by palivizumab and motavizumab. Orange indicates antigenic site IV, targeted by 101 F. (C) Visualization of structurally altered peptides in green (|log₂ FC|>1, moderated t-test, q value <0.01, for all comparisons, n = 4 replicates each for control and treated samples), on one of the subunit of trimeric preRSVF (upper panel) and postRSVF (lower panel) protein structures upon addition of the indicated antibodies. Antigenic sites are colored as in panel (B).