In cell scalaradial interactome profiling using a bio-orthogonal clickable probe

Abstract

A bio-orthogonal click-chemistry procedure was developed to allow the in cell interactome profiling of scalaradial, an anti-inflammatory marine natural product. The results were validated through the application of the classical in vitro chemical proteomics and several bio-physical methods; peroxiredoxins, 14-3-3 isoforms and proteasomes were recognized as main scalaradial targets.

β‐Boswellic acid, a bioactive substance used in food supplements, inhibits protein synthesis by targeting the ribosomal machinery

“…Later on, chemoproteomic results validation and the biological relevance of the main interactions has been assessed through in vitro/in cell assays. [24][25][26][27][28] Experimental Generation of the βBA functional matrix Epoxy-activated Sepharose ™ 6B matrix was swollen with water (200 μl/mg) for 60 min and extensively washed with water. βBA (1 μmol) was diluted in 450 μl of 78% CH3CN/22% NaHCO 3 at pH 8.0 and added to 200 μl of matrix at room temperature for 24 h with continuous shaking.…”

Biomolecular Fishing for Calixarene Partners by a Chemoproteomic Approach

“…[5] Derivative 1a was synthesized (see Scheme S1 in the Supporting Information), purified by HPLC,a nd characterized by 1 Ha nd 13 CNMR spectroscopy and ESI(+ +)M S. [6] It was then linked covalently through its free amino group to solid beads made of CDI agarose activated with 1,1'-carbonyldiimidazole (Scheme 2). Such aspacer is usually required to minimize steric hindrance between the ligand on the matrix and its macromolecular partners during the affinity-purification step.…”

“…Thep rotein mixtures were then purified by SDS-PAGE( see Figure S6);t he entire gel was cut, digested with trypsin, and analyzed by mass spectrometry through nanoflow reversed-phase HPLC MS/ MS ( Figure 1; see also Figure S7). As is usual for this kind of pulldown experiment, [3,5] al arge number of potential partners was found in the raw data. [6] Thei dentified proteins were then compared with those of the free-matrix control experiment (see Table S2), and those present in both experiments were excluded to give as et of potential protein partners (see Table S1).…”

“…[6] Thei dentified proteins were then compared with those of the free-matrix control experiment (see Table S2), and those present in both experiments were excluded to give as et of potential protein partners (see Table S1). As is usual for this kind of pulldown experiment, [3,5] al arge number of potential partners was found in the raw data. This number was strongly reduced by applying af ilter on the Mascot scores and the individual Pep(sig) values,t hus giving al ower, reasonable number of molecules that were potentially interacting with the pAC.…”

In Cell Interactome of Oleocanthal, an Extra Virgin Olive Oil Bioactive Component

“…Then, cells are incubated with the tagged compound to allow its internalization and interactions with the most affine proteins. Furthermore, a mild cell lysis and addition of the suitable complementary reagents launch the click chemistry reaction, also allowing the connection of the small molecule to a solid support [7]. Isolation of this adduct is followed by washing and elution of the pulled-back proteins, subsequently separated by SDS-PAGE and identified by MS-based analysis [7].…”