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首页> 外文期刊>RSC Advances >Immunocapture sample clean-up in determination of low abundant protein biomarkers – a feasibility study of peptide capture by anti-protein antibodies
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Immunocapture sample clean-up in determination of low abundant protein biomarkers – a feasibility study of peptide capture by anti-protein antibodies

机译：测定低丰富蛋白质生物标志物的免疫抑制样品清理 - 抗蛋白抗体肽捕获的可行性研究

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Immunocapture in mass spectrometry based targeted protein analysis using a bottom-up workflow is nowadays mainly performed by target protein extraction using anti-protein antibodies followed by tryptic digestion. Already available monoclonal antibodies (mAbs) which were developed against intact target proteins (anti-protein antibodies) can capture proteotypic epitope containing peptides after tryptic digestion of the sample. In the present paper considerations when developing a method for targeted protein quantitation through capture of epitope containing peptides are discussed and a method applying peptide capture by anti-protein antibodies is compared with conventional immunocapture MS. The model protein used for this purpose was progastrin releasing peptide (ProGRP), a validated low abundant biomarker for Small Cell Lung Cancer with reference values in serum in the pg mL ~(?1) range. A set of mAbs which bind linear epitopes of ProGRP are available, and after a theoretical consideration, three mAbs (E146, E149 and M18) were evaluated for extraction of proteotypic epitope peptides from a complex sample. M18 was the best performing mAb for peptide capture by anti-protein antibodies, matching the LOD (54 pg mL ~(?1) ) and LOQ (181 pg mL ~(?1) ) of the existing conventional immunocapture LC-MS/MS method for determination of ProGRP. Peptide and protein capture using the same mAb were also compared with respect to sample clean-up, and the peptide capture workflow yielded cleaner extracts and therewith less complex chromatograms. Analysis of five patient samples demonstrated that peptide capture by anti-protein antibodies can be used for the determination of various levels of endogenously present ProGRP.
机译：现在，使用抗蛋白质抗体随后进行胰蛋白酶抗体的靶蛋白提取而基于靶向蛋白质分析的基于质谱法的靶向蛋白质分析。已经可用的单克隆抗体（mAb），其用于完整靶蛋白（抗蛋白抗体）可以在样品的胰蛋白酶消解后捕获含有肽的蛋白质表位。在本文的考虑因素中，讨论了通过捕获表位捕获捕获肽的靶向蛋白质定量的方法，并将抗蛋白抗体捕获的方法与常规免疫抑制MS进行比较。用于此目的的模型蛋白质是孕肽（ProgrP），用于小细胞肺癌的验证的低丰富的生物标志物，其中PG mL〜（α1）范围内的血清中的参考值。可以获得一组粘合Progrp的线性表位的MAb，并且在理论考虑之后，评估了三种MAb（E146，E149和M18），用于从复合样品中提取蛋白质表位肽。 M18是通过抗蛋白质抗体捕获的最佳性能mAb，匹配现有常规免疫键入LC-MS / MS的LOD（54pg ml〜（α1））和LOQ（181pg ml〜（α1））分析方法的测定方法。还将使用相同MAb的肽和蛋白质捕获相对于样品清理，并且肽捕获工作流程产生清洁器提取物，从而较差的色谱图。对五个患者样品的分析表明，抗蛋白抗体的肽捕获可用于测定各种内源性的内源性产物。

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