Project description:A spectral library was built for Drosophila melanogaster. The spectral library allows reproducible quantification for thousands of peptides per SWATH-MS analysis.
Proteins from Drosophila melanogaster embryo, adult flies were digested with trypsin using in-gel digestion and the peptides were fractionated by high-pH reverse phase chromatography. HRM peptides were spiked into the peptides mixture and each fraction was injected on a Sciex TripleTOF 6600 mass spectrometer fitted with microflow set-up.
The resulting .wiff files were analysed using MaxQuant and Spectronaut.
Project description:The serine hydrolase (SH) superfamily is perhaps, one of the largest functional enzyme classes in all forms of life, and consists of proteases/peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of the chemical proteomics technique, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlas’ for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, here, we first report a bioinformatics analysis towards the identification and classification of non-redundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the activities of the SH superfamily during various developmental stages and in different adult tissues of Drosophila. Finally, as proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult Drosophila melanogaster, and together, we prospect new research directions, resources and tools that this study can provide to the fly community.