DOSCATs: Double standards for protein quantification.
DOSCATs: Double standards for protein quantification.
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DOI：
10.1038/srep45570
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发表时间：
2017-04-03
影响因子：
4.6
通讯作者：
Beynon RJ
中科院分区：
文献类型：
--
作者：
Bennett RJ;Simpson DM;Holman SW;Ryan S;Brownridge P;Eyers CE;Colyer J;Beynon RJ
绝对蛋白质定量的两种最常见的技术是基于质谱法（MS）或免疫化学技术，如蛋白质印迹法（WB）。蛋白质印迹法最常用于蛋白质鉴定或相对定量，但如果使用适当的校准标准品，也可用于绝对定量。基于MS的技术提供了上级的数据质量和可重复性，但WB为大多数研究人员提供了更高的灵敏度和可访问性。将这两种技术应用于正交量化将是有利的，但工作流程很少重叠。我们描述DOSCATs（双标准conCATamers），新的校准标准的基础上QconCAT技术，统一这些平台。DOSCATs联合收割机将一系列与胰蛋白酶肽串联的表位序列组合在单个人工蛋白中，以产生MS的内部胰蛋白酶肽标准品以及带有多个线性表位的完整蛋白。设计并构建DOSCAT蛋白以定量NF-κB途径的五种蛋白。对于三种靶蛋白，通过MS和WB测量的蛋白质倍数变化和每细胞绝对拷贝值非常一致。这表明DOSCATs可用作多路复用、双重用途的标准品，易于在单个工作流程中部署，支持从MS到WB的无缝定量转换。
The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.