Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
Methods. 2012 Feb;56(2):275-83. doi: 10.1016/j.ymeth.2011.09.021. Epub 2011 Oct 2.
To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.
开发研究蛋白质酪氨酸残基磷酸化的方法是一项重要任务，因为这种蛋白质修饰调节许多细胞功能，并且经常涉及致癌作用。一个最佳的方案包括对酪氨酸磷酸化（pTyr）肽或蛋白质进行富集，然后采用高分辨率的分析方法来鉴定富集的成分。在本方法论文中，我们描述了一种工作策略，即如何将免疫亲和富集（使用抗-pTyr 抗体）与质谱（MS）分析相结合，用于研究 pTyr 蛋白质组。我们详细描述了我们的程序如何用于表征 K562 白血病细胞的 pTyr 蛋白质组。讨论了使用不同的抗-pTyr 抗体、在肽和/或蛋白质水平上进行的富集、富集的合并以及对 MS 鉴定的要求等重要问题。
