Activity based profiling of Phosphoprotein phosphatases in cancer using mass spectrometry-based proteomics

SUMARRY项目 通过激酶抑制剂靶向失调的磷酸化信号传导是一种经证实的策略，也是本领域的焦点。 抗癌治疗的发展。然而，患者反应的可变性和耐药性限制了 并经常导致治疗失败。决定药物疗效的潜在机制是 多方面的，包括药物靶点本身，以及相关的信号通路。例如，突变 或靶向激酶的扩增、相关激酶的功能补偿、激酶的重编程 信号传导和磷酸酶信号传导网络的改变，其拮抗底物磷酸化或 直接调节激酶活性是常见。 在破译癌症激酶组及其对抗癌的反应方面取得了巨大进展 治疗这些研究是由一系列创新技术，包括化学蛋白质组学， 使用固定在珠上的激酶抑制剂和质谱法（MS）的策略。 大多数蛋白质去磷酸化是由磷蛋白磷酸酶（PPPs）进行的。的PPP 家族由九个催化亚基组成，它们与调节亚基和支架亚基结合并组装成 数百种多亚基酶，作为不同的选择性信号实体发挥作用。令人兴奋的是，角色和 PPP在正常和癌组织中的细胞信号传导中的调节开始出现。 虽然激酶组谱分析提供了关于磷酸化反应的全局信息，但是对于磷酸化反应，不存在这样的技术。 因此，我们缺乏对研究不足但同样重要的去磷酸化反应的了解。 在癌症中。为了解决这一能力差距，我们已经建立了一个化学蛋白质组学策略，称为“PIB- 用于所有内源性表达的PPP及其组合的有效亲和捕获、鉴定和定量的“MS”。 相关蛋白质（“PPPome”）在来自有限蛋白质量的细胞的单一质谱分析中， 组织和肿瘤裂解物。在此应用中，我们进一步发展和成熟这项技术，并评估其 在药物治疗干扰后， 和乳腺癌PDX肿瘤模型，以及原发性人乳腺癌肿瘤。我们将评估 在实验室间比较中评估该技术的性能，以实现广泛实施。 潜在影响声明。PPP已成为癌症中的关键信号实体。但目前 目前还没有一种方法可以快速、定量和全面地评估内源性PPPome及其 与细胞应激相关的动态变化，抗癌药物的短期和长期治疗，或 耐药性的发展。PIB-MS是一项高度创新的技术，为客户提供这些功能， 细胞系、疾病的小鼠模型和原发性人类肿瘤的分析。PIB-MS将加速和增强 在基础、转化和临床研究中通过快速和定量的方法对癌症进行分子分析 分析PPP信号传导，其改变，以及其对代偿通路的影响之前，期间和之后 抗癌治疗和耐药性的发展。

PROJECT SUMARRY Targeting dysregulated phosphorylation signaling by kinase inhibitors is a proven strategy and a focus in the development of anti-cancer treatments. However, variability in patient responses and drug resistance limit efficacy and often lead to therapy failure. The underlying mechanisms that determine drug efficacy are multifaceted and include the drug target itself, as well as associated signaling pathways. For instance, mutation or amplification of the targeted kinase, functional compensation by related kinases, reprograming of kinase signaling, and alterations in phosphatase signaling networks that antagonize substrate phosphorylation or directly modulate kinase activity are common. Tremendous progress has been made in deciphering the cancer kinome and its response to anti-cancer treatment. These studies were enabled by an array of innovative technologies, including a chemical proteomics strategy that utilizes kinase inhibitors immobilized on beads and mass spectrometry (MS). The majority of protein dephosphorylation is carried out by phosphoprotein phosphatases (PPPs). The PPP family consists of nine catalytic subunits that bind to regulatory and scaffolding subunits and assemble into hundreds of multi-subunit enzymes and function as distinct, selective signaling entities. Excitingly, the role and regulation of PPPs in cellular signaling in normal and cancerous tissue is beginning to emerge. While kinome profiling provides global information on phosphorylation reactions, no such technology exists for phosphatases; thus, we lack knowledge of the understudied, but equally important, dephosphorylation reaction in cancer. To address this gap in capability, we have established a chemical proteomics strategy called “PIB- MS” for efficient affinity-capture, identification, and quantification of all endogenously expressed PPPs and their associated proteins (“PPPome”) in a single mass spectrometry analysis from limited protein amounts of cell, tissue, and tumor lysate. In this application, we further develop and mature this technology and assess its performance to identify and quantify the PPPome in breast cancer cell lines, upon perturbation by drug treatment, and breast cancer PDX tumor models, and in primary human breast cancer tumors. We will assess the performance of this technology in an inter-laboratory comparison to achieve broad implementation. Statement of Potential Impact. PPPs have emerged as critical signaling entities in cancer. However, currently no approach exists for rapid, quantitative, and comprehensive assessment of the endogenous PPPome and its dynamic changes associated with cellular stress, short- and long-term treatment with anti-cancer drugs, or development of drug resistance. PIB-MS is a highly innovative technology that provides these capabilities for the analysis of cell lines, mouse models of disease, and primary human tumors. PIB-MS will accelerate and enhance the molecular analysis of cancer in basic, translational, and clinical research through rapid and quantitative analysis of PPP signaling, its alterations, and its effects on compensatory pathways before, during, and after anti-cancer treatment and upon development of resistance.