title: Performing protein crosslinking using gas-phase cleavable chemical crosslinkers and liquid chromatography-tandem mass spectrometry.
abstract: In this article, we describe our methods and protocols using collision-induced dissociative chemical crosslinking-tandem mass spectrometry (CID-CXL-MS/MS) analysis and the practical considerations when implementing these reagents and methodology for protein crosslinking studies. The synthesis of our novel chemical crosslinkers is described as well as their use for effectively labeling protein and protein complexes. Several sample preparation methods for liquid chromatography-tandem mass spectrometry are provided including the enrichment of interpeptide crosslinks. For identification of CID-CXL-MS/MS crosslinks, details regarding MS acquisition parameters and the utilization of various mass spectrometers are addressed along with post-data acquisition analysis to identify interpeptide crosslinks. Once the CID-CXL-MS/MS approach is optimized for a protein target or a set of targets, it can be used as a tool for biological research for studying protein structure when integrated with data obtained using other techniques, such as NMR, X-ray crystallography, and cryo-electron microscopy, or extended to the study of protein-protein interactions in physiological environments.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Argo
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States.
authors.forename: Chunxiao
authors.initials: C
authors.lastname: Shi
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States.
authors.forename: Fan
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States. Electronic address: michael_goshe@ncsu.edu.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Goshe
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D012710:Serum Albumin, Bovine
keywords: CID-cleavable crosslinkers; Crosslinking; Gas-phase cleavable crosslinkers; Mass spectrometry; Protein structure; Protein–protein interactions