Progress Toward Proteome-Wide Photo-Crosslinking to Enable Residue-Level Visualization of Protein Structure and Networks in vivo

ABSTRACT: Crosslinking 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 crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have 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-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil symmetrical MS-cleavable linkages upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking in cellulo. These studies elucidate a small portion of E. 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 they will prove useful toward the effort to explore the molecular sociology of the cell.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Escherichia Coli

SUBMITTER: Anneliese Faustino  

LAB HEAD: Stephen Fried

PROVIDER: PXD041148 | Pride | 2023-06-27

REPOSITORIES: Pride