Braitbard Ori, Bishara-Shieban Janette, Glickstein Hava, Kott-Gutkowski Miriam, Pace Umberto, Rund Deborah G, Stein Wilfred D
Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.
MDR Tests Ltd, 28 Pierre Koenig St, Jerusalem, Israel.
Proteome Sci. 2006 Jun 21;4:14. doi: 10.1186/1477-5956-4-14.
We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol.
As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not.
We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment.
我们描述了一种基于酶联免疫吸附测定（ELISA）的检测方法（肽矩阵法），该方法可用于同时鉴定和定量生物样品中的多种蛋白质。首先对生物样品（血液成分、活检组织、培养物或其他）进行裂解，以释放所有蛋白质，无需任何额外的分离步骤。然后用所需的蛋白水解酶对样品中的变性蛋白质进行批量消化。接着使用竞争ELISA方案，通过适当的抗体对消化后的肽段进行检测。
作为其应用实例，本文将肽矩阵法应用于多种细胞系和人淋巴细胞中存在的四种膜蛋白MDR1（P-糖蛋白或ABCB1）、MRP1（ABCC1）、BCRP/MXR（ABCG2）以及钠钾ATP酶α亚基（ATP1A1）的检测。我们表明，使用一系列肽-抗体对，我们能够检测和定量这些蛋白质，并且能够区分表达靶蛋白的细胞系或细胞制剂与不表达的细胞系或细胞制剂。
我们设计了一种基于ELISA的简单蛋白质组学检测方法，该方法能够对细胞或组织样品中的指定蛋白质进行定量，并且在任何实验室中，只需最少的专业设备即可使用。
