Supplementary MaterialsSupporting Info. with spatial and temporal resolution (Scheme 1). Application of CBK1 coupled with quantitative MS enabled the identification of cysteine residues subject to 187389-52-2 oxidation upon growth factor-stimulation in A431 epidermoid carcinoma cells. However, the amount of cysteine residues defined as covalently revised by CBK1 in living cells (~300 cysteines) was less than that of normal IA-alkyne labelling in cell lysates (~1000 cysteines).9, 14 To improve the true amount of cysteine residues determined, we sought to optimize CBK1 to reach at a caged electrophile with improved cysteine labelling properties in living cells. Open up in another window Structure 1 Constructions of caged electrophilic probes for cysteine labelling in living cells. To improve CBK1, derivatives with adjustments towards the linker device (R2) between your electrophile and alkyne, the BK electrophile itself (X), aswell as the caging group (R1) (Structure 1) had been synthesized and examined. Initially, probes CBK2-4 with ether-containing and amide linkers between your NPE-caged BK electrophile as well as the alkyne were synthesized. Labelling efficiencies of CBK2-4 were evaluated in HeLa cell lysates (Figure S1), whereby lysates treated with CBK2-4 (5 M) were irradiated (365 nm, 80 W) on ice, and incubated at 25 C for an hour. A fluorescent rhodamine group was incorporated using CuAAC, followed by SDS-PAGE separation and visualization of protein labelling by in-gel fluorescence. Similar studies were performed to evaluate CBK2-4 protein labelling in living cells upon treatment of cells (250 M to 1 1 mM) for 60 mins prior to irradiation, cell lysis and CD117 in-gel fluorescence analysis (Figure S2). Although CBK2-4 showed cysteine labelling in lysates upon UV irradiation (Figure S1), minimal cysteine labelling 187389-52-2 was observed in living cells, indicating poor membrane permeability of these probes (Figure S2). The BK electrophile was then converted to an iodomethylketone (IK) electrophile, under the hypothesis that IK would be more reactive than BK. An IK electrophile protected with the NPE caging group (CIK1) was synthesized (Scheme 2). Briefly, 6-heptynoic acid (1) was converted to the -hydroxymethyl ketone (11) through acid chloride and diazomethyl ketone intermediates. Next, the ketone group of 11 was transformed to a ketal containing the NPE caging 187389-52-2 group to generate 13. Finally, the hydroxyl group of 13 was triflated, and substituted by iodide to afford CIK1. Protein labelling by CBK1 and CIK1 were evaluated in lysates. Minimal protein labelling was observed without UV irradiation, and the labelling intensity reached a maximum after 3 min of irradiation. Importantly, CIK1 demonstrated higher fluorescence intensity than CBK1 (Figure S3 and S4), which is in accordance with the higher reactivity of the IK electrophile. The ability of iodine to form stronger halogen-bonding interactions with side-chain residues within the protein binding pocket could also be a potential contributor to the observed increase in labelling.18 Open in a separate window Scheme 2 Synthesis of CIK derivatives. Reaction conditions: (a) oxalyl chloride, dichloromethane; (b) (trimethylsilyl)diazomethane, acetonitrile; (c) aqueous sulfuric acid; (d) 1-(o-nitrophenyl)-1,2-ethanediols, pyridinium p-toluenesulfonate, magnesium sulfate, acetonitrile or tetrahydrofuran; (e) trifluoromethanesulfonic anhydride, pyridine, dichloromethane; (f) sodium iodide, acetone. Linker optimization of CIK1 generated CIK-a, a derivative containing an amide linker, which demonstrated the most potent labelling in lysates among the NPE-bearing caged probes (Figure 1A, S6). CIK-a was membrane permeable and displayed robust protein labelling in living cells (Figure S7). Furthermore, in cell-viability assays, CIK-a did not display any apparent cytotoxicity up to 100 M (Figure S8). Protein labelling by 187389-52-2 CIK-a was then evaluated by LC/LC-MS/MS analysis of HeLa cell lysates labelled with CIK-a (100M). Briefly, the MS workflow comprised of incorporation of a chemically cleavable linker (Azo-tag) to CIK-a-labelled proteins using CuAAC, streptavidin enrichment, on-bead trypsin digestion and subsequent release of CIK-labeled peptides for MS analysis.11 Analysis of the resulting fragmentation (MS2) spectra permits identification of the websites of proteins labelling by CIK-a, predicated on the current presence of peptide fragments containing the mass from the IK-probe modification. Regardless of the in-gel fluorescence data, where CIK-a demonstrated higher proteins labelling in accordance with CBK1, 187389-52-2 fewer cysteine residues had been determined for CIK-a in accordance with CBK1 by MS (Desk S1). Further evaluation from the produced MS2 spectra demonstrated the current presence of a predominant fragmentation event in the amide relationship of CIK-a, which precludes following identification from the ensuing peptides predicated on backbone amide fragmentation (Shape S9). These MS data recommended an amide linkage may possibly not be optimal for potential iterations of the caged electrophilic probes. Open up in.