title: Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein-Protein Interactions.
abstract: Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein-protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS(n)). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS(n). Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries.
authors.affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.
authors.forename: Craig B
authors.initials: CB
authors.lastname: Gutierrez
authors.affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.
authors.forename: Clinton
authors.initials: C
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of California , Irvine, California 92697, United States.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Novitsky
authors.affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Huszagh
authors.affiliation: Department of Chemistry, University of California , Irvine, California 92697, United States.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Rychnovsky
authors.affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006736:Horses; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D012451:Safrole / Q000031:analogs & derivatives* / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D003432:Cross-Linking Reagents; D009211:Myoglobin; D010455:Peptides; D012710:Serum Albumin, Bovine; D012451:Safrole; C005746:sulfoxide