title: Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions.
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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Chang Ho
authors.initials: CH
authors.lastname: Sohn
authors.forename: Heather D
authors.initials: HD
authors.lastname: Agnew
authors.forename: J Eugene
authors.initials: JE
authors.lastname: Lee
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Smith
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Gregg
authors.initials: G
authors.lastname: Czerwieniec
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: James R
authors.initials: JR
authors.lastname: Heath
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
authors.forename: J L
authors.initials: JL
authors.lastname: Beauchamp
mesh_terms: D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D025801:Ubiquitin; D001360:Avidin