Disulfide cleavable reagents

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

In another case in which a number of exemplary control experiments were done, Markwell and Fox (1980) crosslinked the outer membranes of enveloped viruses with methyl 3-(p-azidophenyl)dithio]propionimidate. Virus (4 mg protein/ml) was reacted with the imidate (0.1 to 0.5 mM) at 0°C for 30 min at pH 8.5. The reaction was quenched with 50 mM ammonium acetate, 50 mM NEM (30 min, 25 °C), and the vims recovered by centrifugation. After irradiation the crosslinked polypeptides were examined in a two-dimensional SDS-polyacrylamide gel. One complication was that the crosslinking pattern had to be compared with a native pattern of disulfide linkages, and a reagent with a different cleavable crosslink may have been a better choice. As mentioned above, the analysis was simplified by the use of surface labeling. 

Control experiments showed that all the crosslinks were cleavable. No crosslinks were formed if the reagent was omitted, if photolysis was omitted, or if the reagent was first inactivated by treatment with ammonium acetate. These experiments proved that neither monofunctional cross-linking, nor disulfide interchange to give crosslinking in the dark occurred. If the membranes were solubilized in SDS before irradiation no cross-... 

Figure 37 Bifunctional photocross-linking affinity purification reagents with cleavable linkers, (a) Diazirine-modified LacNAc probe with a biotin tag and a cleavable acylsulfonamide linker, (b) Aryl azide Le probe with a biotin tag that contains a cleavable disulfide linker.

Another ICAT reagent, known as HisTag reagent, used to tag Cys residues, has been reported recently [70]. It is a 10-mer derivatized peptide H2N-(His)e-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CONH2 and has a trypsin cleavable site (-Arg-Ala-) that facilitates removal of the Hise-tag during the final digestion step. The thiopyridyl disulfide moiety acts as a thiol reactive group and d4 label is incorporated in Ala-9 residue. 

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