The use of LysargiNase complementary to trypsin in large scale phosphoproteome study for unambiguous phosphosite localization

Understanding of kinase-guided signaling pathways requires identification and analysis of phosphorylation sites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphorylation sites. However, the exact localization of phosphorylation sites which depends on trypsin-centric shotgun phosphoproteomics is often arbitrary and unreliable because of the limited product ion coverage in the MS2 spectra. Therefore, phosphorylation site determination from MS data with a single protease cannot be expected to be comprehensive, regardless of the strategy used for phosphopeptide detection. Here, we have explored the application of LysargiNase which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. Our data demonstrates that the combined application of LysargiNase and trypsin results in higher sequence coverage with more complete ladder sequences which is helpful to pinpoint the precise phosphorylation sites. Therefore, the combined use of these two enzymes will partially overcome the limitations of trypsin-centric phosphoproteomics to achieve a more reliable phosphoproteome and to increase the identification of novel phosphorylation sites.

解析激酶介导的信号通路，需对磷酸化位点开展鉴定与分析。基于质谱（MS）的磷酸化蛋白质组学是一种快速且高灵敏度的高通量磷酸化位点鉴定技术。然而，依赖以胰蛋白酶为核心的鸟枪法磷酸化蛋白质组学策略实现的磷酸化位点精确定位，往往因MS2谱图中产物离子覆盖度有限而具有任意性且可靠性不足。因此，无论采用何种磷酸肽检测策略，仅通过单一蛋白酶从质谱数据中鉴定磷酸化位点，均难以实现全面覆盖。本研究探讨了LysargiNase的应用，该酶此前被报道其酶切特异性与胰蛋白酶呈镜像关系，仅在精氨酸与赖氨酸残基的N端一侧进行切割。本研究数据显示，联合使用LysargiNase与胰蛋白酶可获得更高的序列覆盖度，且能得到更完整的肽段序列梯，这有助于精准定位磷酸化位点。因此，联合使用这两种蛋白酶可部分克服以胰蛋白酶为核心的磷酸化蛋白质组学的局限性，进而获得更可靠的磷酸化蛋白质组，并提升新型磷酸化位点的鉴定数量。