MTS-alkynyldiazirine – A Tag-Transfer Photocrosslinker
Redbrick Molecular sells a collection of Tag-Transfer photocrosslinkers that enable the study of interactions between proteins and other biomolecules. The MTS-alkynyldiazirine product and related compounds were developed by Prof Andy Wilson and Prof Sheena Radford FRS at The University of Leeds.
Chemical cross-linking mass-spectrometry (XL-MS) is a powerful methodology to map interactions between ligands and biomacromolecules and to potentially track changes over time. The Wilson and Radford groups at The University of Leeds have designed four new photo-crosslinking (PXL) reagents that can be used to specifically label free thiols through light-sensitive maleimido or methanethiosulfonate (MTS) groups. Labelled materials can then be studied using mass-spectrometry work flows.
The Leeds MTS and maleimido photocrosslinkers are based on light sensitive diazirines, which offer advantages over alternative crosslinking groups such as benzophenones and aryl nitrenes. The Wilson/Radford reagents react rapidly and indiscriminately upon light radiation and ensure supramolecular connectivity is accurately captured.
These labels can be applied in the structural analyses of protein–protein interactions using PXL-MS by conjugating them to thiol-containing bait peptides/proteins and subsequent crosslinking with partner proteins.
You can now buy the MTS-alkynyldiazirine tag transfer photcrosslinker directly from Redbrick Molecular, but we have also made it available via Fluorochem – http://www.fluorochem.co.uk/Products/Product?code=544255
Photo activated crosslinkers
The methanethiosulfonate or maleimide functional groups are designed to specifically modify cysteine residues that are either naturally occurring or engineered into a “bait” peptide or protein. Direct conjugation of these compounds to cysteine residues makes them suitable labels for proteins and peptides to facilitate structural proteomics studies. The second functional group – the diazirine – is a photoactivated cross-linking group that upon irradiation with 365 nm UV light generates a highly reactive carbene that inserts into proximal functional groups (including C-H, N-H, O-H groups etc) on biomacromolecules which interact with the bait, generating a covalently trapped complex. Reaction of the methanethiosulfonate functional group with cysteine generates a disulfide bond; cleavage of this bond following cross-linking simplifies analysis of crosslinked complexes.
The trifunctional cross-linker contains an extra alkyne group that can be selectively modified using a variety of commercial azides including biotin and various fluorophores. Such modification will further facilitate affinity enrichment of cross-linked proteins using e.g. streptavidin beads.
Customers can purchase the Tag-Transfer compounds individually or as a collection. Click here to see our full range!
- Tag-Transfer compounds and all subsequently labelled proteins should be handled in the dark (i.e. by exclusion of the light) as much as possible and stored at -20°C in amber vials or vials wrapped in aluminium foil.
- The Tag-Transfer products are soluble in DMSO or DMF to at least 10 mg/ml.
- Labelling of cysteine residues should be performed in buffer containing a small amount <10% of DMSO or DMF.
- Reductive removal of the bait initially transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and/or exploited for enrichment, enabling the detection of low abundance crosslinks.
- See the Protocol in the Documents tab for further information.
The Wilson Group is based in the School of Chemistry at The University of Leeds. Its members are also part of the Astbury Centre for Structural Molecular Biology. The group are interested in the application of synthetic molecules to problems in Chemical Biology and Materials Science. Find out more from the group pages.
For further technical details please see the papers published by Prof Wilson and Prof Radford in November 2018 and March 2019 – https://onlinelibrary.wiley.com/doi/10.1002/anie.201809149 ; https://pubs.rsc.org/en/Content/ArticleLanding/2019/RA/C8RA10436K#!divAbstract