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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Optimization of immunoaffinity enrichment and detection: Toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry
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Optimization of immunoaffinity enrichment and detection: Toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry

机译：免疫亲和力富集和检测的优化：通过液相色谱-质谱法全面表征K562细胞的磷酸酪氨酸蛋白质组

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摘要

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.
机译：蛋白酪氨酸残基的磷酸化调节许多细胞功能，并且还被证明与肿瘤发生有关。因此，鉴定细胞的磷酸酪氨酸（pTyr）蛋白质组是非常重要的任务。由于酪氨酸磷酸化仅占整个人类磷酸化蛋白质组的1％左右，因此pTyr蛋白的研究颇具挑战性。在这里，我们报告使用K562细胞作为模型系统的磷酸酪氨酸蛋白质组的优化研究。通过用4G10和PYKD1抗体进行免疫亲和富集，然后进行LC-MS / MS分析，对K562细胞磷酸酪氨酸蛋白质组的主要部分进行了表征。发现对应于342个pTyr蛋白的480个非冗余pTyr肽。 141 pTyr肽未在其他地方进行描述。质谱分析方法被认为是最佳方法，该方法包括对前体离子进行高分辨率FTMS分析并随后检测线性离子阱中的CID片段。对于检测低丰度的pTyr肽，对于一种LC-MS / MS分析，汇集单个免疫亲和力富集至关重要。与4G10抗体相比，首次展示了单克隆PYKD1抗体的富集特性。发现PYKD1对蛋白质富集更有效（分别在肽和蛋白质水平上效率为1.2和5％），而在进行肽富集时4G10表现出更好的结果（15％的效率对蛋白质上的3.6％）。磷酸蛋白质组的基本上不同的亚组被这些抗体富集。该发现与先前的研究一起表明，通过免疫沉淀对pTyr蛋白质组进行全面表征需要使用多种抗体进行亲和力富集。

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