Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, AL, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Aug 1;1228:123815. doi: 10.1016/j.jchromb.2023.123815. Epub 2023 Jul 5.
The extracellular cellular matrix (ECM) maintains tissue structure and regulates signaling functions by continuous degradation and remodeling. Inflammation or other disease conditions activate proteases including matrix metalloproteinases (MMPs) that degrade ECM proteins and in particular generate fragments of collagen and elastin, some of which are biologically active ECM peptides or matrikines. Stepwise degradation of collagen by MMP 8, 9 and prolyl endopeptidase release the matrikine proline-glycine-proline (PGP) and its product acetyl-PGP (AcPGP). These peptides are considered as potential biomarkers and therapeutic targets for many disease conditions such as chronic lung disease, heart disease, and cancer. However, there is no published, validated method for the measurement of PGP and AcPGP in plasma and therefore, we developed a sensitive, selective and reliable, isotope dilution LC-multiple reaction monitoring MS method for their determination in human plasma. The chromatographic separation of PGP and AcPGP was achieved in 3 min using Jupiter column with a gradient consisting of acidified acetonitrile and water at a flow rate of 0.5 ml/min. The limit of detection (LOD) for PGP and AcPGP was 0.01 ng/ml and the limit of quantification (LOQ) was 0.05 ng/ml and 0.1 ng/ml, respectively. Precision and accuracy values for all analytes were within 20 % except for the lowest QC of 0.01 ng/ml. The mean extraction recoveries of these analytes were > 90 % using a Phenomenex Phree cartridge and the matrix effect was < 15 % for all the QCs for PGP and AcPGP except the lowest QC. The stability of PGP and AcPGP was > 90 % in several tested conditions including autosampler use, storage at -80 °C, and after 6 times freeze-thaw cycles. Using this method, we successfully extracted and determined PGP levels in human plasma from healthy and COPD subjects. Therefore, this method is suitable for quantification of these peptides in the clinical setting.
细胞外基质 (ECM) 通过持续的降解和重塑来维持组织结构和调节信号功能。炎症或其他疾病状况会激活包括基质金属蛋白酶 (MMPs) 在内的蛋白酶，这些蛋白酶会降解 ECM 蛋白，特别是产生胶原蛋白和弹性蛋白的片段，其中一些是具有生物活性的 ECM 肽或基质细胞衍生因子。MMP8、9 和脯氨酰内肽酶逐步降解胶原蛋白，释放基质细胞衍生因子脯氨酸-甘氨酸-脯氨酸 (PGP) 和其产物乙酰-PGP (AcPGP)。这些肽被认为是许多疾病状况的潜在生物标志物和治疗靶点，如慢性肺部疾病、心脏病和癌症。然而，目前尚无发表的、经过验证的方法来测量血浆中的 PGP 和 AcPGP，因此，我们开发了一种灵敏、选择性和可靠的、基于 LC-多重反应监测 MS 的同位素稀释法，用于测定人血浆中的 PGP 和 AcPGP。使用 Jupiter 柱，在 0.5ml/min 的流速下，用酸化乙腈和水组成的梯度，在 3 分钟内实现了 PGP 和 AcPGP 的色谱分离。PGP 和 AcPGP 的检测限 (LOD) 分别为 0.01ng/ml 和 0.05ng/ml 和 0.1ng/ml，定量限 (LOQ) 分别为 0.05ng/ml 和 0.1ng/ml。除了最低 QC 浓度为 0.01ng/ml 的外，所有分析物的精密度和准确度值均在 20%以内。使用 Phenomenex Phree 试剂盒，这些分析物的平均提取回收率均大于 90%，除了最低 QC 浓度外，PGP 和 AcPGP 的所有 QC 浓度的基质效应均小于 15%。PGP 和 AcPGP 在包括自动进样器使用、-80°C 储存以及 6 次冻融循环在内的多种测试条件下的稳定性均大于 90%。使用该方法，我们成功地从健康和 COPD 受试者的血浆中提取和测定了 PGP 水平。因此，该方法适用于临床环境中这些肽的定量。
