Department of Pharmacy, University of Oslo.
Department of Pharmacy, University of Oslo;
J Vis Exp. 2023 Jan 13(191). doi: 10.3791/64473.
This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.
本文提出了一个详细描述的方案，用于从干燥样品中高效地提取低丰度蛋白质。这是通过珠基蛋白水解，然后进行特征肽亲和捕获和液相色谱串联质谱（LC-MS/MS）测定来实现的。该方法可应用于传统的纸质卡片干燥样品（如干血斑[DBS]和干血清斑[DSS]），以及使用新型采样方法收集的样品，如体积吸收微采样（VAMS）。除了描述该方法外，本工作还以分步的方式详细介绍了胰蛋白酶珠和抗体包被珠的制备过程。该方法的优点是使用珠子进行高效的蛋白水解，以及使用肽亲和捕获进行选择性的稳健的净化。本研究方案旨在确定低丰度小细胞肺癌（SCLC）生物标志物胃泌素释放肽前体（ProGRP）在干燥血清（DBS 和 VAMS）中的含量。珠粒制备的详细步骤使该工作流程更容易在新的应用或其他实验室中实施。结果表明，采样材料可能会影响结果；在本研究项目中，与 DSS 相比，使用 VAMS 采集的样本信号强度更高。
