Designer Reagents for
Mass Spectrometry-Based Proteomics:
Clickable Cross-Linkers for Elucidation of Protein Structures and
Interactions
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Abstract
We present novel homobifunctional amine-reactive clickable
cross-linkers
(CXLs) for investigation of three-dimensional protein structures and
protein–protein interactions (PPIs). CXLs afford consolidated
advantages not previously available in a simple cross-linker, including
(1) their small size and cationic nature at physiological pH, resulting
in good water solubility and cell-permeability, (2) an alkyne group
for bio-orthogonal conjugation to affinity tags via the click reaction
for enrichment of cross-linked peptides, (3) a nucleophilic displacement
reaction involving the 1,2,3-triazole ring formed in the click reaction,
yielding a lock-mass reporter ion for only clicked peptides, and (4)
higher charge states of cross-linked peptides in the gas-phase for
augmented electron transfer dissociation (ETD) yields. Ubiquitin,
a lysine-abundant protein, is used as a model system to demonstrate
structural studies using CXLs. To validate the sensitivity of our
approach, biotin-azide labeling and subsequent enrichment of cross-linked
peptides are performed for cross-linked ubiquitin digests mixed with
yeast cell lysates. Cross-linked peptides are detected and identified
by collision induced dissociation (CID) and ETD with linear quadrupole
ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and
LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex
systems (e.g., in vivo cross-linking) is illustrated by Western blot
detection of Cul1 complexes including known binders, Cand1 and Skp2,
in HEK 293 cells, confirming good water solubility and cell-permeability