Project description:Diaminoquinazolines represent a privileged scaffold for antimalarial discovery, including use as putative Plasmodium histone lysine methyltransferase inhibitors (BIX-01294). Despite this, robust evidence for their molecular targets, proteome-wide, is lacking. Here we report the design and development of a small-molecule photo-crosslinkable probe to investigate the targets of our diaminoquinazoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labelling of Plasmodium lysates and initial pull-down proteomics experiments identified proteins from different classes enriched by the probe, highlighting the suitability of the developed probe as a valuable tool for protein identification in Plasmodium falciparum.
Project description:Given the pivotal role of Tat in HIV-1 latency, we performed RNA pull-down assays in latently HIV-1-infected J-Lat 10.6 cells using biotinylated RNA probes (including a control RNA probe and a Tat-specific RNA probe) . Cell lysates were incubated with streptavidin agarose resin pre-bound to Tat RNA probes, and the enriched Tat RNA-binding proteins were separated by SDS-PAGE and analyzed by liquid chromatography mass spectrometry (LC-MS/MS)
Project description:Bee venom has been traditionally used in the treatment of inflammatory disorders and is known to contain diverse bioactive peptide components. In this study, we systematically characterized the Apis mellifera venom peptidome and investigated peptides enriched under TRPV1 pull-down conditions with potential inflammatory modulatory activity. Crude venom was fractionated by AKTA chromatography, and individual fractions were evaluated by SDS–PAGE. Protein bands were subjected to in-gel digestion followed by LC–MS/MS protein identification. Fraction F4, which displayed a prominent A280 absorbance peak during AKTA chromatography, was further analyzed by LC–MS/MS–based peptidomics. De novo sequencing results from fraction F4 were used to construct an in-house custom peptide database. Two independent GST pull-down workflows (exploratory and in-house) were performed using GST-TRPV1 and GST controls. Candidate peptides enriched in the GST-TRPV1 pull-down relative to GST controls were identified following background subtraction and peak-area filtering criteria. Selected synthetic peptide candidates were subsequently evaluated using macrophage-based proteomics experiments to assess their potential inflammatory modulatory effects. The deposited dataset includes raw LC–MS/MS files and processed outputs for gel-based protein identification, venom peptidomics, pull-down–associated peptide identification, macrophage proteomics (MaxQuant analysis), and the custom venom peptide FASTA library used for database searches.