Tao W Andy, Wollscheid Bernd, O'Brien Robert, Eng Jimmy K, Li Xiao-jun, Bodenmiller Bernd, Watts Julian D, Hood Leroy, Aebersold Ruedi
The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Nat Methods. 2005 Aug;2(8):591-8. doi: 10.1038/nmeth776.
We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins.
我们提出了一种用于复杂蛋白质混合物中磷酸化位点鉴定及相对定量的稳健通用方法。该方法基于一种新的化学衍生化策略，使用树枝状大分子作为可溶性聚合物载体并结合串联质谱（MS/MS）。在一步反应中，磷酸化肽段在碳二亚胺和咪唑催化的反应中与树枝状大分子共价偶联。修饰后的磷酸肽通过酸水解从树枝状大分子上释放出来，并进行MS/MS分析。当与初始的抗磷酸酪氨酸蛋白免疫沉淀步骤和稳定同位素标记相结合时，在一次实验中，我们鉴定出了T细胞受体（TCR）CD3链基于免疫受体酪氨酸的激活基序（ITAM）内的所有已知酪氨酸磷酸化位点，以及人类T细胞中总共97种酪氨酸磷酸化蛋白及其相互作用伙伴上先前未知的磷酸化位点。还对这些蛋白质中磷酸化的动态变化进行了定量。
