Cross-Linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

The combination of
cross-linking/mass spectrometry (XL-MS) and
ion mobility is still underexplored for conducting protein conformational
and protein–protein interaction studies. We present a method
for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer
that allows separating ions based on their gas-phase mobilities. Cross-linking
was performed with three urea-based MS-cleavable cross-linkers that
deliver distinct fragmentation patterns for cross-linked species upon
collisional activation. The discrimination of cross-linked species
from non-cross-linked peptides was readily performed based on their
collisional cross sections. We demonstrate the general feasibility
of our combined XL-MS/ion mobility approach for three protein systems
of increasing complexity: (i) bovine serum albumin (BSA), (ii) Escherichia coli ribosome, and (iii) HEK293T cell
nuclear lysates. We identified a total of 623 unique cross-linking
sites for BSA, 670 for the E. coli ribosome,
and 1623 unique cross-links for nuclear lysates, corresponding to
1088 intra- and 535 interprotein interactions and yielding 564 distinct
protein–protein interactions. Our results underline the strength
of combining XL-MS with ion mobility not only for deriving three-dimensional
(3D) structures of single proteins but also for performing system-wide
protein interaction studies.