Buhs Sophia, Gerull Helwe, Nollau Peter
Research Institute Children's Cancer Center and Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Building N63, 2nd Floor, 20251, Hamburg, Germany.
Methods Mol Biol. 2017;1555:407-418. doi: 10.1007/978-1-4939-6762-9_23.
Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.
磷酸酪氨酸信号传导在控制许多重要生物学功能（如细胞增殖和凋亡）中发挥着主要作用。因此，解析磷酸酪氨酸依赖性信号传导备受关注，为理解信号转导的生理和病理过程铺平了道路。基于SH2结构域与磷酸酪氨酸残基的特异性结合，我们在此展示了一种通过质谱对酪氨酸磷酸化蛋白进行亲和纯化及后续鉴定的实验流程。结合SH2分析（一种广泛适用于表征细胞提取物中磷酸酪氨酸谱的平台），我们的下拉策略使研究人员目前能够鉴定信号级联反应中被不同SH2结构域差异磷酸化和选择性识别的蛋白质。
