Li Huiyan, Popp Robert, Frohlich Bjorn, Chen Michael X, Borchers Christoph H
University of Victoria-Genome BC Proteomics Centre.
Jewish General Hospital, McGill University.
J Vis Exp. 2017 Aug 18(126):55933. doi: 10.3791/55933.
Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).
质谱（MS）是用于定量复杂样品中蛋白质的最常用技术之一，由于直接检测每个目标分子的质荷比，具有出色的分析特异性。然而，与大多数其他分析技术一样，基于质谱的蛋白质组学倾向于测量高丰度分析物，因此在复杂样品中实现低纳克/毫升或皮克/毫升的检测限具有挑战性，而这正是生物流体（如人血浆）中许多与疾病相关蛋白质的浓度范围。为了辅助检测低丰度分析物，免疫富集已被整合到分析中，以便在质谱测量前浓缩和纯化分析物，从而显著提高分析灵敏度。在这项工作中，提出了免疫基质辅助激光解吸/电离（iMALDI）技术，用于定量生物流体中的蛋白质和肽，该技术基于在磁珠上进行免疫富集，然后在不预先洗脱的情况下进行基质辅助激光解吸/电离质谱（MALDI-MS）测量。抗肽抗体在磁珠上功能化，并与样品孵育。洗涤后，磁珠直接转移到MALDI靶板上，在向磁珠施加基质溶液后，用基质辅助激光解吸/电离飞行时间（MALDI-TOF）仪器测量信号。与其他免疫质谱分析相比，样品制备过程得到简化，并且MALDI测量速度快。整个样品制备通过液体处理系统实现自动化，提高了分析的重现性和通量。在本文中，iMALDI分析用于测定血浆中肽血管紧张素I（Ang I）的浓度，临床上将其用作血浆肾素活性的读数，用于原发性醛固酮增多症（PA）的筛查。
