Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers

Tag‐Convertible Photocrosslinker with Click‐On/Off N‐Acylsulfonamide Linkage for Protein Identification

“…The labeled peptide sequence should provide more direct and reliable information of the target protein. However, the cleavage reaction that proceeds in the Z ‐form requires a rather long irradiation period to be completed as photo‐isomerization reaches equilibrium between E ‐ and Z ‐forms at a ratio of 2:1, and the irradiation period depends on interacting proteins. In this report, the N ‐acylsulfonamide group was incorporated between the crosslinker unit and the ligand moiety, and its well‐regulated cleavage greatly improved overall performance in the PAL‐based target identification from the probe preparation to MS‐based identification of the labeled peptide.…”

“…[15] The orthogonal reactionh as provided excellent advantages in many applications,s uch as probe synthesis, protein modification, and in situ inhibitor synthesis. [16] Recently,p ractical procedures were improved by using trimethoxybenzyl thioesters [17] or 9-fluorenylmethyl (Fm) thioesters [18] rather than labile thioacids.T herefore, the corresponding diazirine derivative 4 (DA thioester) was prepared by coupling (E)-3-(4-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl)-2-methacrylic acid [11] and FmSH. [19] Separately,asulfonyl azide was introduced as the counterpart functional group into as ide chain of a-amino acid (Fmoc-SAzideAlaOH, 3 in Scheme2)d erived from N-Fmoc-cysteine in one-pot synthesis via two-stepr eactions.…”

Hide and seek: Identification and confirmation of small molecule protein targets

“…Other applications of this approach were recently reported. [29][30][31] Linker-free target identification techniques: Savitski et al reported on the proteome-wide profiling of protein thermal stability in the presence of small molecules to determine drug target-engagement in live cells. 32 This method is based on the cellular thermal shift assay (CETSA), which evaluates the melting temperature T m shift of a protein of interest in the presence or absence of a ligand upon thermal denaturation.…”

Development and Leading-Edge Application of Innovative Photoaffinity Labeling

“…68) The ATP probe 59 thus designed successfully created the expected coumarin tag on L-glutamate dehydrogenase protein as well as on a heat shock protein HSP90. The peptide probe 60 for the vacuolar sorting receptor (VSR) of soybean 69) was specifically labeled a recombinant luminal soybean VSR protein (GmVSR) 70) (Fig.…”