Perzanowska Anna, Fatalska Agnieszka, Wojtas Grzegorz, Lewandowicz Andrzej, Michalak Agata, Krasowski Grzegorz, Borchers Christoph H, Dadlez Michal, Domanski Dominik
Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics-Polish Academy of Sciences, Warsaw, Poland.
Mazovian Center of Pulmonary Disease and Tuberculosis Treatment, Otwock, Poland.
Proteomics Clin Appl. 2018 Mar;12(2). doi: 10.1002/prca.201700084. Epub 2018 Feb 7.
The goal of this work was to develop an LC-MRM assay for the quantitative analysis of a set of established and diagnostically important cytokeratin (CK) markers used in cancer diagnosis, prognosis, and therapy monitoring. Second, the potential of this assay in lung cancer diagnosis through pleural effusion (PE) analysis was examined.
A multiplexed MRM assay was developed for 17 CKs and their select caspase-cleaved fragments. Isotope-labeled standard peptides were used for high assay specificity and absolute peptide quantitation; with robust standard-flow LC coupled to a latest-generation triple-quadrupole instrument for high sensitivity. The potential clinical applicability was demonstrated by the analysis of 118 PE samples.
The MRM assay was evaluated for endogenous detection, linearity, precision, upper and lower limits of quantification, selectivity, reproducibility and peptide stability, and is generally applicable to any epithelial cancer study. A set of 118 patients with known pathologies allowed us to define the range of CK levels in clinical PE samples. Specific CKs were able to differentiate cancer-related PEs from those caused by benign ailments. In addition, they allowed to differentiate between PEs from subjects with small cell lung cancer versus non-small cell lung carcinoma, and to further differentiate the latter into its two subtypes, adenocarcinoma and squamous cell carcinoma.
An MRM-based CK assay for carcinoma studies can differentiate between the three lung cancer histological types using less-invasive PE sampling providing potential therapy-guiding information on patients that are inoperable.
本研究的目标是开发一种液相色谱-多反应监测（LC-MRM）检测方法，用于定量分析一组在癌症诊断、预后和治疗监测中已确立且具有重要诊断意义的细胞角蛋白（CK）标志物。其次，通过分析胸腔积液（PE）来检验该检测方法在肺癌诊断中的潜力。
开发了一种针对17种CK及其特定半胱天冬酶切割片段的多重MRM检测方法。使用同位素标记的标准肽来实现高检测特异性和绝对肽定量；将稳健的标准流液相色谱与最新一代三重四极杆仪器联用，以实现高灵敏度。通过对118份PE样本的分析证明了其潜在的临床适用性。
对MRM检测方法进行了内源性检测、线性、精密度、定量上限和下限、选择性、重现性以及肽稳定性的评估，该方法一般适用于任何上皮癌研究。一组118例已知病理情况的患者使我们能够确定临床PE样本中CK水平的范围。特定的CK能够区分癌症相关的PE与良性疾病引起的PE。此外，它们能够区分小细胞肺癌患者与非小细胞肺癌患者的PE，并进一步将后者分为腺癌和鳞状细胞癌两种亚型。
一种基于MRM的用于癌症研究的CK检测方法，可通过侵入性较小的PE采样来区分三种肺癌组织学类型，为无法手术的患者提供潜在的治疗指导信息。
