O'Hayre Morgan, Salanga Catherina L, Dorrestein Pieter C, Handel Tracy M
Skaggs School of Pharmacy and Pharmaceutical Science, University of California, San Diego, La Jolla, California, USA.
Methods Enzymol. 2009;460:331-46. doi: 10.1016/S0076-6879(09)05216-1.
Chemokines induce a number of intracellular signaling pathways by activating second messengers (e.g. calcium) and phosphorylation cascades in order to mediate a myriad of functions including cell migration, survival and proliferation. Although there is some degree of overlap in chemokine receptor-mediated pathway activation, different chemokines will often elicit distinct signaling events. Factors such as cell type, receptor expression levels, G protein availability, and disease state will also influence the signaling response from chemokine-induced receptor activation. Improvements in mass spectrometry, enrichment strategies, and database search programs for identifying phosphopeptides have made phosphoproteomics an accessible biological tool for studying chemokine-induced phosphorylation cascades. Although signaling pathways involved in chemokine-mediated migration have been fairly well characterized, less is known regarding other signaling cascades elicited by chemokines (e.g. to induce proliferation) or the potential for distinct pathway activation in a disease state such as cancer. CXCL12(SDF-1)/CXCR4 signaling has been shown to play an important role in the survival of chronic lymphocytic leukemia (CLL) cells, and thus provides a good system for exploring chemokine signaling, particularly in the interest of survival pathway activation. In this chapter, we describe the use of immobilized metal affinity chromatography (IMAC) phosphopeptide enrichment followed by reversed-phase liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis for exploring CXCL12-mediated signaling in human CLL patient cells.
趋化因子通过激活第二信使（如钙离子）和磷酸化级联反应来诱导多种细胞内信号通路，从而介导包括细胞迁移、存活和增殖在内的众多功能。尽管趋化因子受体介导的通路激活存在一定程度的重叠，但不同的趋化因子通常会引发不同的信号事件。细胞类型、受体表达水平、G蛋白可用性和疾病状态等因素也会影响趋化因子诱导的受体激活所产生的信号反应。用于鉴定磷酸肽的质谱分析、富集策略和数据库搜索程序的改进，使得磷酸化蛋白质组学成为研究趋化因子诱导的磷酸化级联反应的一种可利用的生物学工具。尽管趋化因子介导的迁移所涉及的信号通路已得到相当充分的表征，但对于趋化因子引发的其他信号级联反应（如诱导增殖）或在癌症等疾病状态下不同通路激活的可能性了解较少。CXCL12（SDF-1）/CXCR4信号通路已被证明在慢性淋巴细胞白血病（CLL）细胞的存活中起重要作用，因此为探索趋化因子信号传导，特别是在存活通路激活方面，提供了一个良好的系统。在本章中，我们描述了使用固定化金属亲和色谱（IMAC）磷酸肽富集，随后进行反相液相色谱和串联质谱（LC-MS/MS）分析，以探索人CLL患者细胞中CXCL12介导的信号传导。
