Pinto Gabriella, Illiano Anna, Serpico Stefania, Maurelli Fabio, Scaglione Elena, Colicchio Roberta, Vitiello Mariateresa, Varelli Marco, Salvatore Paola, Amoresano Angela
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.
National Institute of Biostructures and Biosystems, Interuniversity Consortium, Via dei Carpegna, 19, 00165, Rome, Italy.
Anal Bioanal Chem. 2025 Jun 25. doi: 10.1007/s00216-025-05950-9.
Inflammation has been considered a pivotal player in the most severe forms of respiratory infections like COVID-19, which induces a significant alteration of inflammatory cytokines called cytokine storm. Above the immune system, abnormalities in coagulation parameters have been observed in COVID-19-affected subjects displaying an increased risk of a blood clot causing thromboembolic events. Currently, clinical methods for monitoring infections rely on serological tests that detect the antibody response using enzyme-linked immunosorbent assays (ELISA). While ELISA offers high sensitivity, it is limited by complex procedures, significant matrix interference, and antibody cross-reactivity. This study aims to develop a mass spectrometry (MS)-based analytical method to support clinical investigations into the host response to infection. The use of S-Trap column digestion in a short time overcomes the limitations of traditional bottom-up proteomics protocols by significantly reducing processing time and eliminating the need for a desalting step. A targeted tandem MS approach using multiple reaction monitoring (MRM) ion mode was established to simultaneously quantify a panel of approximately 60 proteins associated with the inflammatory response and coagulation cascade. Among the proteins analyzed, 90% exhibited a good instrumental response, with 63% showing significant dysregulation in COVID-19 patients compared to controls, enabling the identification of key protein changes linked to the host's infection response. The proposed method leverages the enhanced sensitivity and selectivity of the multiplexed LC-MRM/MS technique, made possible by triple quadrupole mass spectrometers. These instruments effectively filter precursor and product ions, allowing specific monitoring of fragmentation patterns unique to each peptide sequence. By combining a rapid digestion protocol with a multiplexed LC-MRM/MS approach, this method offers a valuable solution for clinical laboratories aiming for high-specificity, high-performance analyses within a shortened analysis time.
炎症被认为是 COVID-19 等最严重形式的呼吸道感染中的关键因素，它会引发称为细胞因子风暴的炎症细胞因子的显著变化。除免疫系统外，在受 COVID-19 影响的受试者中还观察到凝血参数异常，这些受试者发生血栓形成导致血栓栓塞事件的风险增加。目前，监测感染的临床方法依赖于血清学检测，即使用酶联免疫吸附测定（ELISA）来检测抗体反应。虽然 ELISA 具有高灵敏度，但它受到操作程序复杂、显著的基质干扰和抗体交叉反应性的限制。本研究旨在开发一种基于质谱（MS）的分析方法，以支持对宿主感染反应的临床研究。在短时间内使用 S-Trap 柱消化克服了传统自下而上蛋白质组学方案的局限性，通过显著减少处理时间并消除脱盐步骤的需要。建立了一种使用多反应监测（MRM）离子模式的靶向串联质谱方法，以同时定量一组约 60 种与炎症反应和凝血级联相关的蛋白质。在所分析的蛋白质中，90%表现出良好的仪器响应，与对照组相比，63%在 COVID-19 患者中表现出显著失调，从而能够识别与宿主感染反应相关的关键蛋白质变化。所提出的方法利用了三重四极杆质谱仪实现的多重液相色谱-多反应监测/质谱（LC-MRM/MS）技术增强的灵敏度和选择性。这些仪器有效地过滤前体离子和产物离子，允许对每个肽序列独特的碎片模式进行特异性监测。通过将快速消化方案与多重 LC-MRM/MS 方法相结合，该方法为临床实验室提供了一种有价值的解决方案，旨在在缩短的分析时间内进行高特异性、高性能分析。
