Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium.
Laboratory of Immunobiology, Department of Microbiology, Immunology and Transplantation, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium.
Front Immunol. 2021 Mar 11;12:644725. doi: 10.3389/fimmu.2021.644725. eCollection 2021.
With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.
通过 ELISA 可以检测生物样本中免疫反应性分子的整体。对于因蛋白水解而激活、增强或抑制的生物分子，需要其他技术来研究生物学。在这里，我们开发了一种将免疫吸附样品制备和纳米液相色谱-串联质谱（nano-LC-MS/MS）相结合的方法，用于进行蛋白形式分析（ISTAMPA），并将其应用于糖基化趋化因子 CXCL8。CXCL8 是最强大的人类趋化因子，具有中性粒细胞趋化和激活特性，由于其易于发生特异性蛋白水解修饰，因此存在不同的 NH2-末端蛋白形式。特定的蛋白形式显示出高达 30 倍的增强活性。基于免疫吸附离子阱自上而下质谱法的蛋白形式分析方法可用于同时检测和定量生物样品中的全长 CXCL8(1-77)、伸长的 CXCL8(-2-77)和所有天然存在的截断 CXCL8 形式。我们首次证明了慢性关节炎症患者滑液中 CXCL8 的特异性蛋白水解激活，并强调了样本采集和处理的重要性。
