Integrating proteomics with electrochemistry for identifying kinase biomarkers.
Integrating proteomics with electrochemistry for identifying kinase biomarkers.
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DOI：
10.1039/c5sc00560d
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发表时间：
2015-08-01
期刊：
影响因子：
8.4
通讯作者：
Yitzchaik S
中科院分区：
文献类型：
--
作者：
Amit E;Obena R;Wang YT;Zhuravel R;Reyes AJF;Elbaz S;Rotem D;Porath D;Friedler A;Chen YJ;Yitzchaik S
我们提出了一种综合的方法，用于高度敏感地识别和验证底物特异性激酶作为癌症生物标记物。我们提出了一种综合的方法，用于高度敏感地识别和验证底物特异性激酶作为癌症生物标记物。我们的方法结合了用于从患者组织中高通量发现与癌症相关的生物标记物的磷酸蛋白质组学和用于灵敏验证的阻抗式激酶活性生物传感器。以非小细胞肺癌(NSCLC)为概念验证研究，对一对癌组织及癌旁正常组织进行了无标记定量磷蛋白质组学分析，发现198个磷酸蛋白在NSCLC中过度磷酸化。在差异调节的磷酸化位点中，最显著的变化是肝癌衍生生长因子(HDGF)蛋白中的S165残基。因此，选择HDGF作为电化学研究的模型体系。进一步的基于基序的分析表明，细胞外信号调节的激酶1/2(ERK1/2)最有可能是相应的激酶。为了验证激酶-底物对，将与HDGF磷酸化位点相对应的致密堆积的多肽单分子层连接到金电极上。通过电化学阻抗谱(EIS)和表面粗糙度分析检测ERK2对单层膜的磷酸化和碱性磷酸酶(AP)对单层膜的脱磷作用。与其他定量检测激酶浓度的方法相比，这种无标记的电化学方法具有超高灵敏度和高特异性的优点，可用于检测癌症相关的激酶-底物对。随着多个激酶-底物生物标记物对的实施，我们期待这种集成的方法成为发现和验证磷酸化介导的生物标记物的高通量平台。
We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. Our approach combines phosphoproteomics for high throughput cancer-related biomarker discovery from patient tissues and an impedimetric kinase activity biosensor for sensitive validation. Using non-small-cell lung cancer (NSCLC) as a proof-of-concept study, label-free quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed 198 phosphoproteins that are over-phosphorylated in NSCLC. Among the differentially regulated phosphorylation sites, the most significant alteration was in residue S165 in the Hepatoma Derived Growth Factor (HDGF) protein. Hence, HDGF was selected as a model system for the electrochemical studies. Further motif-based analysis of this altered phosphorylation site revealed that extracellular-signal-regulated kinase 1/2 (ERK1/2) are most likely to be the corresponding kinases. For validation of the kinase–substrate pair, densely packed peptide monolayers corresponding to the HDGF phosphorylation site were coupled to a gold electrode. Phosphorylation of the monolayer by ERK2 and dephosphorylation by alkaline phosphatase (AP) were detected by electrochemical impedance spectroscopy (EIS) and surface roughness analysis. Compared to other methods for quantification of kinase concentration, this label-free electrochemical assay offers the advantages of ultra-sensitivity as well as higher specificity for the detection of cancer-related kinase–substrate pair. With implementation of multiple kinase–substrate biomarker pairs, we expect this integrated approach to become a high throughput platform for discovery and validation of phosphorylation-mediated biomarkers.
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影响因子：
14.9
作者：
Hornbeck PV;Kornhauser JM;Tkachev S;Zhang B;Skrzypek E;Murray B;Latham V;Sullivan M
通讯作者：
Sullivan M
影响因子：
7
作者：
Han, Chia-Li;Chien, Chih-Wei;Chen, Yu-Ju
通讯作者：
Chen, Yu-Ju
影响因子：
6.6
作者：
Jorcin, JB;Orazem, ME;Tribollet, B
通讯作者：
Tribollet, B
影响因子：
12.8
作者：
Bam, Rakesh;Ling, Wen;Khan, Sharmin;Pennisi, Angela;Venkateshaiah, Sathisha Upparahalli;Li, Xin;van Rhee, Frits;Usmani, Saad;Barlogie, Bart;Shaughnessy, John;Epstein, Joshua;Yaccoby, Shmuel
通讯作者：
Yaccoby, Shmuel
影响因子：
11.2
作者：
Buonato JM;Lazzara MJ
通讯作者：
Lazzara MJ