Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions
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.
© 2012 American Chemical Society. Published In Issue March 20, 2012; Article ASAP March 05, 2012; Just Accepted Manuscript February 17, 2012; Received: October 11, 2011; Accepted: February 17, 2012. C.H.S. and H.D.A. contributed equally to this work. This work was supported by the National Science Foundation through grants CHE-0416381 (J.L.B.) and U54CA151819-01 (J.R.H.), and the Beckman Institute at Caltech (J.L.B., M.J.S., S.H. and R.L.J.G.) and the Betty and Gordon Moore Foundation (S.H. and R.L.J.G.). J.A.L acknowledges support from the NIH (RR 20004). Computational resources for quantum chemical calculations were kindly provided by the Materials and Process Simulation Center at Caltech. We are also grateful to Bert Lai for technical assistance with the CD experiments. C.H.S. acknowledges a fellowship for a graduate study abroad from the Kwanjeong Educational Foundation. J.E.L. was supported by the Ruth L. Kirschstein NRSA fellowship from the NIH (CA138126).
Supplemental Material - ac202637n_si_001.pdf
Accepted Version - nihms-361514.pdf