Leibniz-Institute for Molecular Pharmacology, Berlin, Germany.
Proteomics. 2009 Dec;9(23):5288-95. doi: 10.1002/pmic.200900294.
The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S-tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C- or N-terminally to the S3S-tag. After retroviral transduction of a vector containing S3S-tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP-tagged protein close to endogenous levels. The combination of a cleavable S-tag and a Strep-tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture-based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co-purification of the known interaction partner Src kinase-associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF-H1 and the adaptor protein DOCK2 corroborates a link between ADAP-mediated integrin regulation and the cytoskeleton.
细胞功能模块化单元的鉴定是蛋白质组学研究的主要目标。蛋白质复合物代表着重要的功能构建块，而解析其组成仍然是一个主要的挑战。在这里，我们设计了一种新的串联亲和纯化（TAP）标签（称为 S3S-tag），用于分离蛋白质复合物。具体来说，调节整合素黏附的免疫细胞蛋白 ADAP 要么 C 端要么 N 端融合到 S3S-tag 上。在含有 S3S-tagged ADAP 和内部核糖体进入位点编码增强型绿色荧光蛋白（eGFP）的载体转导后，根据 eGFP 表达对 Jurkat T 细胞进行分选，并进一步选择表达接近内源性水平的 TAP-tagged 蛋白。可切割 S-tag 和 Strep-tag II 的组合允许分离 ADAP 和相关蛋白。随后，进行基于细胞培养的稳定同位素标记氨基酸的质谱分析，以鉴定潜在的特异性相互作用伙伴。已知相互作用伙伴 Src 激酶相关磷酸蛋白 55kDa 的共纯化表明了我们方法的有效性，而 ENA/VASP 家族成员 EVL、鸟嘌呤核苷酸交换因子 GEF-H1 和衔接蛋白 DOCK2 的鉴定证实了 ADAP 介导的整合素调节与细胞骨架之间的联系。
