Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS Cleavable Cross-Linker

Cross-linking
combined with mass spectrometry (MS) has evolved
as an alternative strategy in structural biology for characterizing
three-dimensional structures of protein assemblies and for mapping
protein–protein interactions. Here, we describe an integrated
workflow for an automated identification of cross-linked products
that is based on the use of a tandem mass spectrometry (MS/MS) cleavable
cross-linker (containing a 1,3-bis-(4-oxo-butyl)-urea group, BuUrBu)
generating characteristic doublet patterns upon fragmentation. We
evaluate different fragmentation methods available on an Orbitrap
Fusion mass spectrometer for three proteins and an E. coli cell lysate. An updated version of the dedicated software tool MeroX
was employed for a fully automated identification of cross-links.
The strength of our cleavable cross-linker is that characteristic
patterns of the cross-linker as well as backbone fragments of the
connected peptides are already observed at the MS/MS level, eliminating
the need for conducting MS3 or sequential CID (collision-induced
dissociation)- and ETD (electron transfer dissociation)-MS/MS experiments.
This makes our strategy applicable to a broad range of mass spectrometers
with MS/MS capabilities. For purified proteins and protein complexes,
our workflow using CID-MS/MS acquisition performs with high confidence,
scoring cross-links at 0.5% false discovery rate (FDR). The cross-links
provide structural insights into the intrinsically disordered tetrameric
tumor suppressor protein p53. As a time-consuming manual inspection
of cross-linking data is not required, our workflow will pave the
way for making the cross-linking/MS approach a routine technique for
structural proteomics studies.