Progress toward Proteome-Wide Photo-Cross-Linking to Enable Residue-Level Visualization of Protein Structures and Networks In Vivo

Cross-linking
mass spectrometry (XL-MS) is emerging as a method
at the crossroads of structural and cellular biology, uniquely capable
of identifying protein–protein interactions with residue-level
resolution and on the proteome-wide scale. With the development of
cross-linkers that can form linkages inside cells and easily cleave
during fragmentation on the mass spectrometer (MS-cleavable cross-links),
it has become increasingly facile to identify contacts between any
two proteins in complex samples, including in live cells or tissues.
Photo-cross-linkers possess the advantages of high temporal resolution
and high reactivity, thereby engaging all residue-types (rather than
just lysine); nevertheless, photo-cross-linkers have not enjoyed widespread
use and are yet to be employed for proteome-wide studies because their
products are challenging to identify. Here, we demonstrate the synthesis
and application of two heterobifunctional photo-cross-linkers that
feature diazirines and N-hydroxy-succinimidyl carbamate
groups, the latter of which unveil doubly fissile MS-cleavable linkages
upon acyl transfer to protein targets. Moreover, these cross-linkers
demonstrate high water-solubility and cell-permeability. Using these
compounds, we demonstrate the feasibility of proteome-wide photo-cross-linking in cellulo. These studies elucidate a small portion of Escherichia coli’s interaction network, albeit
with residue-level resolution. With further optimization, these methods
will enable the detection of protein quinary interaction networks
in their native environment at residue-level resolution, and we expect
that they will prove useful toward the effort to explore the molecular
sociology of the cell.