Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide e-(?-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins.
Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide e-(?-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins.
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液相色谱-三重四极杆质谱方法的开发和验证，用于测定人尿液中作为转谷氨酰胺酶 2 交联蛋白生物标志物的异肽 e-(β-谷氨酰) 赖氨酸。
DOI：
10.1016/j.chroma.2023.464002
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Dejager L
中科院分区：
文献类型：
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作者：
Dejager L
在不同的疾病状态下，测定转谷氨酰胺酶2活性所催化的蛋白质交联度仍然是一个重大的挑战。同肽(ε-(γ-谷氨酰基)赖氨酸可以在蛋白质内或蛋白质之间形成异构键，对其进行量化的能力具有显著的分析和临床潜力，可作为人体尿液等生物体液中的生物标志物。转谷氨酰胺酶2活性升高与许多疾病有关，如纤维化。以前发表的方法都是基于经典的氨基酸分析，然而它们需要复杂的多酶消化才能实现完全的蛋白质消化，同时保持异肽的交联键不变。这些方法需要高水平的酶，这会污染分析并改变消化的动态。氨基酸分析检测也缺乏选择性，特别是在预计交联度相对于背景蛋白水平较低的情况下。我们已经系统地解决了这些挑战，通过优化尿液中蛋白质的沉淀，使用创新的固定化酶技术，允许高效消化而不受酶污染，以及基于多反应监测的LC-MS/MS检测。对该方法的分析性能进行了验证，结果表明，该方法对人尿中ε-(γ-谷氨酰赖氨酸的灵敏度为0.1ngL/m L，精密度小于20%，且具有选择性，因为没有观察到可能对分析产生不利影响的干扰。因此，这种方法在检测和定量ε-(γ-谷氨酰赖氨酸的能力方面取得了重大进展。
Determination of the levels of protein cross-linking catalysed by the activity of transglutaminase 2 in various disease states has remained a significant challenge. The ability to quantify the isopeptide ε-(γ-glutamyl) lysine, which can form as a heterogeneous bond within or between proteins has significant analytical and clinical potential as a biomarker in biofluids such as human urine. Increased transglutaminase 2 activity is associated with a number of diseases, such as fibrosis. Previously published methods have been based on classical amino acid analysis, however they require a complex multi-enzyme digestion in order to achieve complete protein digestion, whilst leaving the isopeptide cross link intact. These methods require high levels of enzymes, which contaminate the analysis and alter the dynamics of digestion. The amino acid analysis detection also lacked selectivity, especially where the levels of crosslink are expected to be low relative to the background protein levels. We have systematically addressed these challenges, by optimising the precipitation of the protein in urine, the use of innovative immobilised enzyme technology, which allows for efficient digestion without enzyme contamination and LC-MS/MS detection based on multiple reaction monitoring. This method was validated for its analytical performance characteristics, showing the method has a sensitivity of 0.1 ng/mL of ε-(γ-glutamyl) lysine in human urine with precision of less than 20% CV, and is selective as no interferences were observed that may adversely affect the analysis. As such this approach represents a significant advance in the ability to detect and quantify ε-(γ-glutamyl) lysine.