13 research outputs found
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Synthesis of Photoreactive Mass Spec Cleavable Crosslinkers to Study Protein-Protein Interactions
The following document describes the synthesis and preliminary testing of three novel CID-cleavable crosslinking agents for the study of protein-protein interactions. These crosslinkers contain a sulfoxide group that can be cleaved inside of the mass spectrometer at a lower energy than the peptide backbone, allowing for a series of tandem experiments to identify peptide fragments. Three crosslinkers of varying length containing a lysine-reactive N-hydroxysuccinimide ester and a photoreactive diazirine ring were developed. One crosslinker was functionalized with an alkyne to allow for affinity purification with biotin. Two of the crosslinkers have been found to successfully form crosslinks within synthetic peptides and bovine serum albumin, and experiments are currently ongoing with the third crosslinking agent
Recommended from our members
Synthesis of Photoreactive Mass Spec Cleavable Crosslinkers to Study Protein-Protein Interactions
The following document describes the synthesis and preliminary testing of three novel CID-cleavable crosslinking agents for the study of protein-protein interactions. These crosslinkers contain a sulfoxide group that can be cleaved inside of the mass spectrometer at a lower energy than the peptide backbone, allowing for a series of tandem experiments to identify peptide fragments. Three crosslinkers of varying length containing a lysine-reactive N-hydroxysuccinimide ester and a photoreactive diazirine ring were developed. One crosslinker was functionalized with an alkyne to allow for affinity purification with biotin. Two of the crosslinkers have been found to successfully form crosslinks within synthetic peptides and bovine serum albumin, and experiments are currently ongoing with the third crosslinking agent
Plan of Renovation Of Greely Institute, Main Street, Cumberland, Maine, 1972
https://digitalmaine.com/cumberland_plans/1019/thumbnail.jp
Plan of Renovation Of Greely Institute, Main Street, Cumberland, Maine, 1972
https://digitalmaine.com/cumberland_plans/1019/thumbnail.jp
Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers.
Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies