Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K8-98, Richland, WA, 99354, USA.
Institute of Precision Medicine of Weill Cornell Medical College and New York Presbyterian Hospital, New York, NY, USA.
J Transl Med. 2017 Aug 15;15(1):175. doi: 10.1186/s12967-017-1276-7.
Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region with multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations.
To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations.
Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification ranged from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present, it is unknown if this also affects the biological activity of the SPOP protein.
In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, providing significant potential in biomarker development for prostate cancer.
斑点型 POZ 蛋白（SPOP）是一种 E3 泛素连接酶衔接蛋白，作为一种潜在的肿瘤抑制因子发挥作用，约 10%的人类前列腺癌中存在 SPOP 突变。然而，突变的 SPOP 蛋白是否可以作为前列腺癌早期检测、诊断、预后或靶向治疗的生物标志物仍不清楚。此外，SPOP 突变位点分布在具有多个赖氨酸残基的相对较短区域，这对 SPOP 突变的自上而下蛋白质组学分析提出了重大挑战。
为了解决这个问题，开发了 PRISM（高压、高分辨率分离与智能选择和多路复用）-SRM（选择反应监测）质谱测定法来定量野生型 SPOP 蛋白和 11 种前列腺癌衍生的 SPOP 突变。
尽管由于氨基酸序列限制存在固有局限性，但使用 Arg-C 消化开发的所有 PRISM-SRM 测定法均表现出至少两个数量级的线性动态范围，定量限范围为细胞裂解物中总蛋白的 0.1 至 1 fmol/μg。将这些 SRM 测定法应用于分析表达前列腺癌中最常见的三种 SPOP 突变（Y87N、F102C 或 F133V）的 HEK293T 细胞，导致在相应的阳性细胞系中能够可靠地检测到所有三种 SPOP 突变，但在阴性细胞系中则无法检测到。与 F102C 和 Y87N 突变相比，F133V 突变和野生型 SPOP 的表达水平要低得多；然而，目前尚不清楚这是否也会影响 SPOP 蛋白的生物学活性。
总之，PRISM-SRM 能够在细胞裂解物中进行突变 SPOP 蛋白的多重、异构体特异性检测，为前列腺癌的生物标志物开发提供了重要潜力。
