Faria Morse, Halquist Matthew S, Yuan Moucun, Mylott William, Jenkins Rand G, Karnes H Thomas
Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, 23298, USA.
Chromatographic Sciences, PPD, 2244 Dabney Road, Richmond, VA, 23230, USA.
Biomed Chromatogr. 2015 Nov;29(11):1780-2. doi: 10.1002/bmc.3471. Epub 2015 Apr 27.
A stable isotope-labeled signature peptide, whose sequence corresponds to the human osteopontin (hOPN) specific antibody epitope, was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity-coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well from 150 to 4500 ng and analysis was carried out with internal standards added before and after the immunocapture step. The immunocapture variability ranged from -80.9 to 77.0% when the IS was added after immunocapture and from -37.5 to 20.3% when the internal standard was added before immunocapture. The lower variability demonstrates the ability of stable labeled isotope internal standard peptide to compensate for variation during immunocapture.
一种稳定同位素标记的特征肽，其序列与人骨桥蛋白（hOPN）特异性抗体表位相对应，被评估作为内标，以补偿在通过免疫亲和偶联液相色谱 - 串联质谱法定量hOPN过程中的免疫捕获变异性。通过将每孔抗体量从150 ng变化至4500 ng来诱导免疫捕获变异性，并在免疫捕获步骤之前和之后添加内标进行分析。当内标在免疫捕获后添加时，免疫捕获变异性范围为-80.9%至77.0%，而当内标在免疫捕获前添加时，变异性范围为-37.5%至20.3%。较低的变异性证明了稳定标记的同位素内标肽补偿免疫捕获过程中变异的能力。
