Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further-tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for This method using standard peptides and proteins. Peptides and proteins were cross-linked and their fragmentation characteristics were analyzed using CID and ETD tandem mass spectrometry. Two sequential cleavages unambiguously identified cross-linked peptides. In addition, the labeling efficiency of the new cross-linker was evaluated in macrophage immune cells after stimulation with the microbial ligand lipopolysaccharide and subsequent pulldown experiments with biotin-avidin affinity chromatography. We believe this strategy will help advance insights into the structural biology and systems biology of cell signaling.
机构:
Univ Cambridge, Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Cambridge CB2 0RE, EnglandUniv Cambridge, Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Cambridge CB2 0RE, England
机构:Univ British Columbia, Biomed Res Ctr, Dept Chem, Vancouver, BC V6T 1Z3, Canada
Sutherland, Brent W.
Toews, Judy
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机构:
Univ British Columbia, Biomed Res Ctr, Dept Chem, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, Biomed Res Ctr, Dept Chem, Vancouver, BC V6T 1Z3, Canada
Toews, Judy
Kast, Juergen
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机构:
Univ British Columbia, Biomed Res Ctr, Dept Chem, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, Biomed Res Ctr, Dept Chem, Vancouver, BC V6T 1Z3, Canada
Kast, Juergen
JOURNAL OF MASS SPECTROMETRY,
2008,
43
(06):
: 699
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715