Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic colon lesions.
Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic colon lesions.
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DOI：
10.1002/pmic.201200430
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发表时间：
2013-05
期刊：
影响因子：
3.4
通讯作者：
Rosenberg, Daniel W.
中科院分区：
文献类型：
--
作者：
Drew, David A.;Devers, Thomas;Horelik, Nicole;Yang, Shi;O'Brien, Michael;Wu, Rong;Rosenberg, Daniel W.
关键词：
在假定的癌前病变，异常隐窝病灶（ACF）中，已确定致癌激活导致结肠粘膜内的过度增殖性病变。通过原位高清晰度放大色素内镜检查，在筛选受试者的结直肠中确定了172例ACF，确定了KRAS和BRAF突变状态。如前所述，根据组织学（锯齿状vs.扩张）对病变进行分层。然而，由于其有限的大小，检查这些致癌突变的下游信号传导结果在技术上是不可行的。我们结合了紫外-红外（UV/IR）显微切割与超灵敏的纳米流体蛋白质组学免疫测定（NIA），使翻译后修饰的丝裂原活化蛋白激酶（MAPK）的准确定量总蛋白裂解物中分离的过度增殖的隐窝和邻近的正常粘膜。使用这种方法，在从<200个细胞中回收的含有少至16 ng总蛋白的样品中定量细胞外受体激酶（ERK）-1和-2的单一和双重（活化）磷酸化亚型的水平。ERK激活是这些早期病变中观察到的增生的原因，但不直接依赖于KRAS和/或BRAF突变状态。这项研究描述了一种灵敏的纳米流体平台的新用途，以测量癌基因驱动的蛋白质组学变化的微小病变，并强调了这种方法的优势，经典的荧光化学为基础的分析。
Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS and BRAF mutation status was determined in 172 ACF identified in the colorectum of screening subjects by in situ high-definition, magnifying chromoendoscopy. Lesions were stratified according to histology (serrated vs. distended) as previously described. Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet-infrared (UV/IR) microdissection with an ultrasensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of post-translational modifications to mitogen-activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)-1 and -2 were quantified in samples containing as little as 16-ng of total protein recovered from <200 cells. ERK activation is responsible for observed hyperplasia found in these early lesions, but is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene-driven proteomic changes in diminutive lesions and highlights the advantage of this approach over classical immunohistochemistry-based analyses.
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