One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination

IF 1.9 3区 化学 Q3 BIOCHEMICAL RESEARCH METHODS
Ago Mrsa, Greta Nardini, Trine Grønhaug Halvorsen, Bernd Thiede, Léon Reubsaet
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Abstract

This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte. The antibody-based paper samplers were prepared by functionalizing paper discs (6 mm) through a simple reaction using divinyl sulfone (DVS). After DVS activation, the paper discs were incubated with E27 hCG mAbs, followed by 0.05% tween/phosphate buffer saline to block the surface. After sample application and drying, the discs only needed to be washed before tryptic digestion and finally analysed on a nanoliquid chromatography–tandem mass spectrometry system. The finished DVS-mAbs samplers could selectively capture hCG (100 ng/mL) from human serum, with a recovery of 50%. Sample clean-up reduced the number of identified proteins from 132 to 82 before and after wash, respectively, with a 70% reduction in serum albumin signal while still retaining hCG on the sampler during the washing protocol. An evaluation of the samplers revealed excellent linearity (R2 = 0.9995) for hCG in serum with relative standard deviations below 15%. This work has presented the first ever reported paper samplers immobilized with antibodies utilizing DVS chemistry, showing promise in the future of paper-based sampling.

Abstract Image

在基于液相色谱-串联质谱的靶向蛋白质测定中,对纸张进行一步功能化和简化抗体固定,以便当场从干燥血清中进行免疫捕获。
这项工作旨在简化和改进将单克隆抗体(mAbs)共价结合到滤纸上的过程,以便用于干血斑采样,从而即时捕获蛋白质生物标记物,进行有针对性的蛋白质测定。将必要的免疫捕获样品制备步骤纳入初始采样阶段,既节省了时间,又减少了工作量。生物标记物人绒毛膜促性腺激素(hCG)被用作模型分析物。抗体纸采样器是通过使用二乙烯基砜(DVS)的简单反应对纸盘(6 毫米)进行功能化而制备的。DVS 活化后,用 E27 hCG mAbs 对纸盘进行孵育,然后用 0.05% 的吐温/磷酸盐缓冲盐水对纸盘表面进行阻断。样品涂抹和干燥后,只需在胰蛋白酶消化前对纸盘进行清洗,最后在纳米液相色谱-串联质谱系统上进行分析。完成的 DVS-mAbs 采样器可选择性地捕获人血清中的 hCG(100 ng/mL),回收率达 50%。样品清理后,鉴定出的蛋白质数量从洗涤前后的 132 个分别减少到 82 个,血清白蛋白信号减少了 70%,同时在洗涤过程中采样器上仍能保留 hCG。对采样器的评估显示,血清中 hCG 的线性度非常好(R2 = 0.9995),相对标准偏差低于 15%。这项工作首次报道了利用 DVS 化学方法固定抗体的纸质采样器,显示了纸质采样的未来前景。
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来源期刊
Journal of Mass Spectrometry
Journal of Mass Spectrometry 化学-光谱学
CiteScore
5.10
自引率
0.00%
发文量
84
审稿时长
1.5 months
期刊介绍: The Journal of Mass Spectrometry publishes papers on a broad range of topics of interest to scientists working in both fundamental and applied areas involving the study of gaseous ions. The aim of JMS is to serve the scientific community with information provided and arranged to help senior investigators to better stay abreast of new discoveries and studies in their own field, to make them aware of events and developments in associated fields, and to provide students and newcomers the basic tools with which to learn fundamental and applied aspects of mass spectrometry.
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