Project description:In 2022 a vineyard in southeastern Pennsylvania was found to be infested with black widow spiders. Despite attempts to harvest proteins from the surface of an aluminum baseball bat, proteomic coverage was quite poor. In 2023, a mature female was temporarily stunned by the shockwave of an aluminum ballbat on concrete in a blow that altered the shape of both objects. This example of Latrodectus was transferred into a 15 mL falcon tube by a mass spectrometrist who made very undignified noises while making this transfer. The tube was rapidly sealed and the tube was placed at -20C before being transferred to -80C for storage. After the project was declined by multiple people in the laboratory a new student in the lab accepted this as his first proteomics project. The spider was carefully removed from the tube but the freeze/thaw process only allowed the separation of 2 distinct sections, titled "body" and "legs". These were homogenized in 4 fractions per section in a bead mill using 30 second pulses in 1x S-Trap lysis buffer. The resulting lysate was clarified by brief centrifugation and the lysates were digested using the S-Trap Mini spin columns following vendor protocols including the reduction and alkylation of cysteines with DTT and iodoacetamide. Peptides concentrations were determined by colorimetric assay (Pierce) and 400 nanograms of each were loaded onto EvoTips for analysis using a 30SPD method. A diaPASEF method on a TIMSTOF Flex instrument was used for analysis. Resulting samples were processed in SpectroNaut 18 using a combination of Latrodectus and Spider fastas from UniProt, as described in the manuscript.
