Department of Biomedical and Chemical Engineering and Sciences, Florida Institute of Technology, Melbourne, FL, USA.
Department of Biochemistry and Molecular Genetics, Midwestern University, Glendale, AZ, USA.
Methods Mol Biol. 2021;2219:119-135. doi: 10.1007/978-1-0716-0974-3_7.
The signaling mechanisms controlling internal calcium release at fertilization in animals are still largely unknown. Echinoderms, such as the sea star Patiria miniata, produce abundant and easily accessible sperm and eggs. In addition, eggs are naturally synchronized at the same cell cycle stage, collectively making these animals an attractive model to study the signaling proteins controlling fertilization. However, the lack of antibodies to identify proteins in this model system has slowed progress in identifying key signaling molecules. With the advances in mass spectrometry, we present a method for identifying tyrosine phosphorylated proteins binding to GST-tagged SH2 domains in sea star cell lysates for downstream mass spectrometry analysis.
动物受精时控制内部钙释放的信号机制在很大程度上仍然未知。棘皮动物，如海星 Patiria miniata，产生丰富且易于获取的精子和卵子。此外，卵子在同一细胞周期阶段自然同步，这使得这些动物成为研究控制受精的信号蛋白的理想模型。然而，由于缺乏识别该模型系统中蛋白质的抗体，鉴定关键信号分子的进展受到了阻碍。随着质谱技术的进步，我们提出了一种在海星细胞裂解物中鉴定与 GST 标记的 SH2 结构域结合的酪氨酸磷酸化蛋白的方法，用于下游质谱分析。
