Drosophila cSPH35 cSPH242 cleavage site identification

The purified pro-cSPHs (ca. 10 μg) were incubated with PAP3 (1.0 μg) for 1 h at 37 ℃. The mixtures and negative controls of the pro-cSPHs were denatured in urea and digested with chymotrypsin or V8 protease overnight at 37 ℃ (Zhang et al., 2014). The resulting peptides were desalted using Omix C18 affinity media, dried, and dissolved in mobile phase A (0.1% formic acid in H2O). The samples were loaded onto an Acclaim PepMap RSLC C18 column (75 μm × 50 cm, Thermo Fisher) for data-dependent LC-MS/MS analysis as described previously (Cao et al., 2020). Peptides in each sample were separated via a gradient of 0–35% mobile phase B (0.1% formic acid in 80% AcCN) developed over 120 min (Jin et al., 2022). The survey scans were followed by both HCD and CID collisional MS/MS events triggered from the hypothetical peptide ions, with scanning of collisional fragment at 15,000 resolutions in the Orbitrap sector. For identifying the PAP3 cleavage sites in cSPH242 and cSPH35, a database combining D. melanogaster pro-cSPH242 and pro-cSPH35 and background proteins from M. sexta, human, and insect cells were constructed, and peptide spectrum matches were reviewed in Byonic to detect peptides not cut by nonspecific proteases. The details of peak area calculation were described previously to quantify characteristic peptides released by PAP3 and chymotrypsin/V8 protease (Jin et al, 2022).