Photocleavable peptide-oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS.
Photocleavable peptide-oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS.
复制标题
DOI：
10.1039/c2mb25163a
复制
发表时间：
2012-09
影响因子：
--
通讯作者：
Kron SJ
中科院分区：
文献类型：
--
作者：
Zhou G;Khan F;Dai Q;Sylvester JE;Kron SJ
对细胞蛋白酪氨酸激酶活性进行高度平行、定量分析的可靠方法可能提供破译致癌信号、发现新的靶向药物、诊断癌症和监测患者至关重要的工具。在这里，我们描述了一种新的蛋白激酶分析的原理证明，它有可能回答这些挑战。MALDI-TOF质谱学为多肽磷酸化的无标记多路分析提供了理想的工具，但与均相分析和复杂样品的匹配性较差。因此，我们将一个共同的寡核苷酸标签连接到多个多肽底物上，通过对连接到聚乙二醇钝化的超顺磁性微粒上的互补序列进行退火，有效地捕获了溶液相激酶反应。为了实现可逆连接，我们开发了一种新型的双功能交联剂，可以简单有效地制备可光裂解的多肽-寡核苷酸连接物。洗涤污染物和光释放后，MALDI-TOF分析得到每个肽的相对磷酸化，具有很高的灵敏度和特异性。验证了杂交法，当三种肽底物-寡核苷酸结合物与酪氨酸激酶c-Abl和ATP混合时，我们很容易观察到它们的差异磷酸化，并测量了Abl激酶抑制剂伊马替尼的共同IC50。这一新的分析方法能够以多种形式分析蛋白激酶活性，从而能够并行筛选针对多个激酶的抑制剂，这是药物发现和预测性诊断的重要能力。
Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with potential to answer these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptide-oligonucleotide conjugates. After washing away contaminants and photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate-oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC50 for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics.
登录
查看更多内容
影响因子：
4.7
作者：
Christie RJ;Anderson DJ;Grainger DW
通讯作者：
Grainger DW
影响因子：
3.5
作者：
Akita, Shoji;Umezawa, Naoki;Higuchi, Tsunehiko
通讯作者：
Higuchi, Tsunehiko
影响因子：
--
作者：
Jia, Yong;Quinn, Christopher M;Talanian, Robert V
通讯作者：
Talanian, Robert V
影响因子：
2.9
作者：
Kumar EA;Charvet CD;Lokesh GL;Natarajan A
通讯作者：
Natarajan A
影响因子：
46.9
作者：
Kubota, Kazuishi;Anjum, Rana;Yu, Yonghao;Kunz, Ryan C.;Andersen, Jannik N.;Kraus, Manfred;Keilhack, Heike;Nagashima, Kumiko;Krauss, Stefan;Paweletz, Cloud;Hendrickson, Ronald C.;Feldman, Adam S.;Wu, Chin-Lee;Rush, John;Villen, Judit;Gygi, Steven P.
通讯作者：
Gygi, Steven P.