Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor
Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor
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DOI：
10.1016/j.jasms.2006.09.004
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Niidome, Takuro
中科院分区：
文献类型：
--
作者：
Kang, Jeong-Hun;Katayama, Yoshiki;Niidome, Takuro
一种基于质谱的新型测定系统使用质量标记的底物肽探针来确定蛋白激酶活性，用于肿瘤的诊断。合成了两种肽探针（H 型和 D 型），其中含有相同的蛋白激酶 C (PKC) 底物肽序列。由于 D 型探针的乙酰基中掺入了氘，因此两种探针的分子量不同。制备正常组织和肿瘤组织的裂解物并分别与H型和D型肽探针反应。通过计算每个肽探针的磷酸化比率（从两种反应溶液混合后的质谱峰强度获得），可以简单直接地比较正常组织和肿瘤组织的PKC活性。 H型肽探针与肿瘤组织裂解液（B16黑色素瘤）反应的磷酸化率比D型肽探针与正常皮肤组织裂解液反应的磷酸化率高出三倍以上。这些结果表明，使用质量标签技术检测蛋白激酶活性的新型测定系统可以是一种简单而有用的方法来分析细胞或组织裂解物样品的蛋白激酶活性，并且可以应用于肿瘤的诊断。
A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.