Department of Pharmaceutical chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, NO-0316 Oslo, Norway.
Department of Pharmaceutical chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, NO-0316 Oslo, Norway.
Talanta. 2019 Apr 1;195:764-770. doi: 10.1016/j.talanta.2018.12.013. Epub 2018 Dec 7.
A novel sampling concept for mass spectrometric bottom-up targeted protein analysis is here demonstrated with polymeric sampling spots integrated with instant immunocapture for analysis of dried matrix spots. The polymers 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) and pHEMA-Tosyl for covalent attachment of antibodies where investigated alongside with adsorption on non-treated filter paper. From performance characterization, the pHEMA-VDM had the best performance. The sampling spots demonstrated fast and easy sampling and preparation of human serum spiked with the biomarker human chorionic gonadotropin. The sampling spots enabled a detection limit of 1 ng/mL (26.4 pM) within a five point concentration curve from 1 ng/mL to 20 ng/mL (R = 0.97). The detection limit was demonstrated to be two times lower than previously demonstrated with standard DMPK-C sampling cards. A five point concentration curve from 100 ng/mL to 2000 ng/mL was also investigated (R = 0.998). Intra day precision was within 16% and 23% for concentration range 1 ng/mL to 20 ng/mL and 100 ng/mL to 2000 ng/mL, respectively. Inter day precision was within 20%. Accuracy was determined to 10% and 11% for 2.5 ng/mL and 20 ng/mL, respectively. The sampling spots were also demonstrated in a realistic setting where serum samples from two confirmed patients with testicular germ cell cancer were analyzed. These analyses confirmed an elevated hCG content in the sera of 418.5 ± 4.2 ng/mL and 21 ± 0.02 ng/mL hCG for patient one and two respectively.
一种新型的用于质谱法自上而下靶向蛋白质分析的采样概念，这里展示了与即时免疫捕获集成的聚合物采样点，用于分析干燥基质点。研究了用于共价连接抗体的 2-羟乙基甲基丙烯酸酯-共-2-乙烯基-4,4-二甲基恶唑啉（pHEMA-VDM）和 pHEMA-Tosyl 聚合物，以及吸附在未经处理的滤纸上的聚合物。从性能表征来看，pHEMA-VDM 的性能最佳。这些采样点可快速、轻松地对人血清进行采样和制备，其中加入了生物标志物人绒毛膜促性腺激素。采样点能够在从 1ng/mL 到 20ng/mL（R=0.97）的五点浓度曲线中检测到 1ng/mL（26.4pM）的下限。与先前使用标准 DMPK-C 采样卡的检测相比，检测下限降低了两倍。还研究了从 100ng/mL 到 2000ng/mL 的五点浓度曲线（R=0.998）。在 1ng/mL 到 20ng/mL 和 100ng/mL 到 2000ng/mL 的浓度范围内，日内精密度分别为 16%和 23%。日间精密度为 20%。对于 2.5ng/mL 和 20ng/mL，准确度分别确定为 10%和 11%。在实际环境中也对采样点进行了测试，分析了两名经证实的睾丸生殖细胞癌患者的血清样本。这些分析证实了患者一和二的血清 hCG 含量分别为 418.5±4.2ng/mL 和 21±0.02ng/mL。
