Pharma Research Basel, F. Hoffmann-La Roche, CH-4070 Basel, Switzerland.
Anal Biochem. 2013 May 15;436(2):127-36. doi: 10.1016/j.ab.2013.01.012. Epub 2013 Feb 8.
Elastin is one of the major extracellular matrix proteins associated with connective tissue. Its degradation leads to the liberation of the unique amino acids desmosine and isodesmosine. These have shown utility as biomarkers of elastin breakdown for disease progression, patient stratification, and drug efficacy. So far, the quantitation of desmosines in plasma is hampered by complex sample preparation. Here we demonstrate an improved and simplified procedure for detecting both free and total desmosines. The method is based on spiking with a deuterium-labeled desmosine standard, ethanol precipitation, propionylation, high-performance liquid chromatography (HPLC) separation, and selected reaction monitoring (SRM) mass spectrometry. The performance of the assay is illustrated by comparing the levels of free and total desmosines in normal healthy plasma and those from patients diagnosed with chronic obstructive pulmonary disease (COPD). A conserved ratio of 1:3 for free to total desmosine was found. The determination of free desmosine has higher accuracy than that of total desmosine; therefore, it is the method of choice when plasma volume is limiting. Finally, we show that the plasma desmosine concentration correlates with age and body mass index.
弹性蛋白是与结缔组织相关的主要细胞外基质蛋白之一。其降解导致独特的氨基酸表位-desmosine 和异表位-desmosine 的释放。这些已被证明可作为弹性蛋白分解的生物标志物，用于疾病进展、患者分层和药物疗效的评估。到目前为止，由于复杂的样品制备，血浆中表位-desmosine 的定量受到阻碍。在这里，我们展示了一种改进和简化的方法来检测游离和总表位-desmosine。该方法基于氘标记的表位-desmosine 标准品的掺入、乙醇沉淀、丙酰化、高效液相色谱 (HPLC) 分离和选择反应监测 (SRM) 质谱。通过比较正常健康血浆和慢性阻塞性肺疾病 (COPD) 患者的游离和总表位-desmosine 水平，说明了该测定方法的性能。发现游离表位-desmosine 与总表位-desmosine 的比例为 1:3。游离表位-desmosine 的测定具有比总表位-desmosine 更高的准确性；因此，当血浆体积有限时，它是首选方法。最后，我们表明血浆表位-desmosine 浓度与年龄和体重指数相关。
