title: Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking.
abstract: 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.
authors.forename: Jayanta K
authors.initials: JK
authors.lastname: Chakrabarty
authors.forename: Aishwarya G
authors.initials: AG
authors.lastname: Naik
authors.affiliation: Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH , Research Triangle Park, North Carolina 27709, United States.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Fessler
authors.affiliation: Laboratory of Bioanalysis, Washington State University , Pullman, Washington 98195, United States.
authors.forename: Gerhard R
authors.initials: GR
authors.lastname: Munske
authors.forename: Saiful M
authors.initials: SM
authors.lastname: Chowdhury
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006835:Hydrazones / Q000737:chemistry; D051379:Mice; D009496:Neurotensin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D000067996:RAW 264.7 Cells; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D006835:Hydrazones; D010455:Peptides; D011506:Proteins; D013388:Succinimides; D025801:Ubiquitin; D012710:Serum Albumin, Bovine; D009496:Neurotensin