van der Burgt Yuri E M, Siliakus Kasper M, Cobbaert Christa M, Ruhaak L Renee
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
J Proteome Res. 2020 Jul 2;19(7):2708-2716. doi: 10.1021/acs.jproteome.0c00050. Epub 2020 Mar 18.
Elevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity values of 20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in overdiagnosis and unnecessary interventions. Previous exploratory studies have indicated that the glycosylation of PSA potentially leads to improved PCa diagnosis based on qualitative analyses. However, the applied methods are not suited for a quantitative evaluation or implementation in a medical laboratory. Therefore, in this proof-of-principle study, we have evaluated the use of hydrophilic interaction liquid chromatography (HILIC) in combination with targeted quantitative mass spectrometry for the sialic acid linkage-specific analysis of PSA glyco-proteoforms based on either trypsin or ArgC peptides. The efficiency of PSA proteolysis was optimized as well as the glycopeptide separation conditions (buffer type, strength, and pH). The HILIC-based analysis of PSA glyco-proteoforms presented here has the potential for the clinical validation of patient cohorts. The method shows the feasibility of the use of a HILIC stationary phase for the separation of isomeric glycopeptides to detect specific glyco-proteoforms. This is the first step toward the development and evaluation of PSA glyco-proteoforms for use in a clinical chemistry setting aiming for improved PCa diagnosis or screening.
体液中血清前列腺特异性抗原（PSA）水平升高可能提示前列腺癌（PCa），但应注意其临床性能相当差。在临界值为4.1 ng/mL时，特异性和敏感性值分别为20%和94%，这会导致过度诊断和不必要的干预。先前的探索性研究表明，基于定性分析，PSA的糖基化可能有助于改善PCa诊断。然而，所应用的方法不适合在医学实验室进行定量评估或应用。因此，在本原理验证研究中，我们评估了亲水相互作用液相色谱（HILIC）结合靶向定量质谱用于基于胰蛋白酶或ArgC肽对PSA糖蛋白亚型进行唾液酸连接特异性分析的情况。优化了PSA蛋白水解的效率以及糖肽分离条件（缓冲液类型、强度和pH值）。本文介绍的基于HILIC的PSA糖蛋白亚型分析具有对患者队列进行临床验证的潜力。该方法显示了使用HILIC固定相分离异构体糖肽以检测特定糖蛋白亚型的可行性。这是朝着开发和评估用于临床化学环境以改善PCa诊断或筛查的PSA糖蛋白亚型迈出的第一步。
