Human Health Therapeutics Research Center, National Research Council, Halifax, Nova Scotia, United Kingdom.
Nova Scotia Health Authority, Division of Medical Oncology, Halifax, Nova Scotia, United Kingdom.
PLoS One. 2023 Jun 29;18(6):e0281491. doi: 10.1371/journal.pone.0281491. eCollection 2023.
Osteopontin (OPN) is a secreted glycophosphoprotein that derives its name from its high abundance in bone and secretion by osteoblasts. It is also secreted by a number of immune cells and, therefore, is present in human plasma at nanogram per millilitre levels where it affects cell adhesion and motility. OPN is involved in several normal physiological processes; however, OPN dyregulation leads to overexpression by tumor cells leading to immune evasion and increased metastasis. Plasma OPN is primarily measured by enzyme-linked immunosorbent assay (ELISA). However, due to the complexity of the various OPN isoforms, conflicting results have been obtained on the use of OPN as a biomarker even in the same disease condition. These discrepant results may result from the difficulty in comparing ELISA results obtained with different antibodies that target unique OPN epitopes. Mass spectrometry can be used to quantify proteins in plasma and, by targeting OPN regions that do not bear post-translational modifications, may provide more consistent quantification. However, the low (ng/mL) levels in plasma present a significant analytical challenge. In order to develop a sensitive assay for plasma OPN, we explored a single-step precipitation method using a recently developed spin-tube format. Quantification was performed using isotope-dilution mass spectrometry. The concentration detection limit of this assay was 39 ± 15 ng/mL. The assay was applied to the analysis of plasma OPN in metastatic breast cancer patients, where levels from 17 to 53 ng/mL were detected. The sensitivity of the method is higher than previously published methods and sufficient for OPN detection in large, high grade tumors but still requires improvement in sensitivity to be widely applicable.
骨桥蛋白（OPN）是一种分泌型糖磷蛋白，因其在骨骼中含量丰富且由成骨细胞分泌而得名。它也由许多免疫细胞分泌，因此，在人类血浆中以纳克/毫升的水平存在，影响细胞黏附和运动。OPN 参与了几种正常的生理过程；然而，OPN 的失调导致肿瘤细胞过度表达，导致免疫逃逸和转移增加。血浆 OPN 主要通过酶联免疫吸附测定（ELISA）进行测量。然而，由于各种 OPN 同工型的复杂性，即使在相同的疾病条件下，使用 OPN 作为生物标志物也得到了相互矛盾的结果。这些不一致的结果可能是由于比较针对独特 OPN 表位的不同抗体获得的 ELISA 结果存在困难。质谱可用于定量血浆中的蛋白质，并且通过针对不带有翻译后修饰的 OPN 区域，可能提供更一致的定量。然而，血浆中的低（ng/mL）水平存在显著的分析挑战。为了开发用于检测血浆 OPN 的灵敏测定法，我们探索了一种使用最近开发的旋转管格式的单步沉淀方法。使用同位素稀释质谱法进行定量。该测定法的浓度检测限为 39±15ng/mL。该测定法应用于转移性乳腺癌患者的血浆 OPN 分析，检测到 17 至 53ng/mL 的水平。该方法的灵敏度高于以前发表的方法，足以检测大、高级别肿瘤中的 OPN，但仍需要提高灵敏度以广泛应用。
