Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis
Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis
复制标题
用于深度酪氨酸磷酸化蛋白质组分析的双相亲和色谱方法
DOI：
10.1021/acs.analchem.6b04288
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发表时间：
2017-02-21
影响因子：
7.4
通讯作者：
Ye, Mingliang
中科院分区：
文献类型：
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作者：
Deng, Zhenzhen;Dong, Mingming;Ye, Mingliang
酪氨酸磷酸化（pTyr）对于正常生理学是重要的，并且涉及许多人类疾病，特别是癌症。pTyr位点的鉴定对于剖析信号传导途径和理解疾病病理是至关重要的。然而，与丝氨酸/苏氨酸磷酸化（pSer/pThr）相比，由于pTyr的丰度较低，因此在蛋白质组水平上分析pTyr更具挑战性。在这里，我们开发了一种双相亲和层析方法，其中Src SH 2 superbinder与NeutrAvidin亲和层析相结合，用于酪氨酸磷酸化蛋白质组分析。通过使用竞争性洗脱剂biotin-pYEEI，该策略可以区分高亲和力磷酸酪氨酸肽和低亲和力磷酸酪氨酸肽，而过量的竞争性试剂可以通过使用NeutrAvidin琼脂糖树脂在集成的尖端系统中容易地去除。通过分析Jurkat细胞的酪氨酸磷酸化蛋白质组证明了该系统的优异性能，从该蛋白质组中鉴定出3，480个独特的pTyr位点。用于深度Tyr磷酸化蛋白质组分析的双相亲和色谱法快速、灵敏、稳健且具有成本效益。它广泛适用于与酪氨酸激酶信号转导相关的酪氨酸磷酸化蛋白质组的整体分析。
Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.