Easy peptm mini ms sample prep kit

Wildtype and homozygous (n = 6 per group) ventricular samples, previously collected from embryos at E15.5, were prepared and labelled using solutions provided in EasyPepTM mini MS sample prep kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. For data analysis, the MS and MS/MS scans were searched against the Uniprot database using Proteome Discoverer 2.2 (Thermo Fisher Scientific, Waltham, MA, USA) with 5% false discovery rate (FDR) criteria. Multiple test correction was performed using the Benjamini and Hochberg test (Perseus software [21 (link)]). Data were further analysed through the use of Ingenuity pathway analysis (IPA, QIAGEN Inc, Germantown, MD, USA), https://digitalinsights.qiagen.com/IPA, (accessed on 22 February 2023) [22 (link)]). Detailed information is provided in the Supplementary Materials.
The proteomics data underlying this article are available in PRIDE [https://www.ebi.ac.uk/pride/archive/, (accessed on 3 January 2023)], and can be accessed with identifier PXD039226.

The EasyPep^TM Mini MS Sample Prep Kit was utilised as per the manufacturer’s protocol (Thermo Fisher Scientific, North Ryde, NSW, Australia) for chemical and enzymatic sample processing, using 100 μg of protein samples. Using lysis buffer (#1863441, Thermo Fisher Scientific, North Ryde, NSW, Australia), the final volume was adjusted to 100 μL in a microcentrifuge tube. The reduction and alkylation solutions were added at a volume of 50 μL each, gently mixed, and further incubated at 95 °C for 10 min using a heat block. The samples were cooled at RT, and 50 μL of the reconstituted trypsin/lys-C protease mixture was added to each sample, then incubated (with shaking) at 37 °C for 3 h. Following incubation, 50 μL of digestion stop solution was added and gently mixed. Peptide clean-up columns were implemented to remove both hydrophobic and hydrophilic contaminants. Following the clean-up, clean peptide samples were then dried using a vacuum centrifuge and resuspended in 100 μL 0.1% formic acid in water for the LCMS analysis, and gently placed in maximum recovery sample vials (Waters Corp., Milford, MA, USA).

Proteomic analysis was performed on 3 biological replicates of SAS cells culture media-derived extracellular vesicles (EVs) treated or not with 25 ppm of BPA, with different times and doses of neutron irradiation (0 min with 0 Gy, 10 min with 1.9 Gy and 60 min with 11.3 Gy) and with two-time points of EVs isolation (6 h and 24 h post-irradiation) (n = 3, in different conditions). For each EV sample, 100 µg of protein mixture was reduced/alkylated and enzymatically digested using Easy Pep^TM Mini MS Sample Prep Kit (Thermo Fisher Scientific, Rockford, IL, USA). Following the kit protocol, in less than 3 h, peptides were generated for each examined condition, cleaned up to prepare detergent-free samples and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) for nLC-hrMS/MS analysis.