Protein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential. For breast cancer treatment, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method. Data are available via ProteomeXchange with identifiers PXD044655 and PXD046169.

中文翻译：

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用于临床蛋白质组学的激酶抑制剂下拉测定 (KiP)

蛋白激酶在癌症中经常失调和/或突变，是治疗的重要靶点。准确的量化至关重要。对于乳腺癌治疗，蛋白激酶 ERBB2 的鉴定和定量对于治疗决策至关重要。虽然免疫组织化学 (IHC) 是当前的临床诊断方法，但它只是半定量的。与 IHC 不同，基于质谱的蛋白质组学提供定量分析，可用于同时准确评估数百种激酶。富集不太丰富的激酶靶点进行定量，同时去除干扰蛋白，提高了灵敏度，从而促进更有效的下游分析。因此，多种激酶抑制剂被用作激酶抑制剂下拉 (KiP) 测定的捕获基质，旨在尽可能广泛地分析人类蛋白激酶组。优化的检测最初在 16 个患者来源的异种移植模型 (PDX) 中进行了评估，其中 KiP 鉴定了多种差异表达和生物学相关的激酶。根据这些分析，开发了一种优化的单次平行反应监测（PRM）方法来提高定量保真度。然后，将 PRM KiP 方法重新应用于人类癌症芯针活检中典型产量的少量蛋白质。针对 100 种激酶的初始原型概括了从全面的蛋白质组学和转录组学分析中获得的 PDX 模型的内在亚型。随后通过 KiP-PRM 对 Luminal 和 HER2 富集 OCT 冷冻患者活检进行分析，也按亚型进行聚类。最后，开发了稳定同位素标记的肽标准品来定义原型临床方法。数据可通过 ProteomeXchange 获得，标识符为 PXD044655 和 PXD046169。