Drosophila cSPH35 cSPH242 cleavage site identification

The purified pro-cSPHs (<em>ca</em>. 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 <em>D. melanogaster</em> pro-cSPH242 and pro-cSPH35 and background proteins from <em>M. sexta</em>, 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).

将纯化的前体cSPHs（pro-cSPHs，约10 μg）与PAP3（1.0 μg）在37 ℃下孵育1小时。将上述反应混合液及前体cSPHs阴性对照经尿素变性后，于37 ℃下用胰凝乳蛋白酶（chymotrypsin）或V8蛋白酶（V8 protease）过夜酶解（Zhang等，2014）。所得肽段经Omix C18亲和介质（Omix C18 affinity media）脱盐、冻干，并用流动相A（含0.1%甲酸的去离子水）复溶。将样品加载至Acclaim PepMap RSLC C18色谱柱（75 μm × 50 cm，赛默飞世尔科技（Thermo Fisher）），按照此前报道的方法进行数据依赖性LC-MS/MS分析（Cao等，2020）。每份样品中的肽段经120分钟的梯度洗脱分离，流动相B为含0.1%甲酸的80%乙腈（AcCN）溶液，洗脱梯度为0%~35%（Jin等，2022）。一级全扫描后，以假设的肽段离子触发高能碰撞解离（HCD）和碰撞诱导解离（CID）两种模式的二级质谱扫描，在轨道阱（Orbitrap）质量分析器中以15000的分辨率采集碰撞片段离子数据。为鉴定cSPH242和cSPH35中的PAP3剪切位点，构建了包含黑腹果蝇（D. melanogaster）pro-cSPH242、pro-cSPH35以及烟草天蛾（M. sexta）、人类和昆虫细胞背景蛋白的数据库，并通过Byonic软件对肽段谱匹配结果进行审核，以识别未被非特异性蛋白酶切割的肽段。峰面积计算的详细步骤此前已有报道，用于定量PAP3及胰凝乳蛋白酶/V8蛋白酶释放的特征性肽段（Jin等，2022）。