School of Pharmacy , Nanjing Medical University , 818 Tian Yuan East Road , Nanjing , Jiangsu , China , 211166.
China State Key Laboratory of Reproductive Medicine , Nanjing , China 210029.
Anal Chem. 2018 Mar 6;90(5):3058-3066. doi: 10.1021/acs.analchem.7b02890. Epub 2018 Feb 15.
Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.
C 末端 p53 异构体 α、β 和 γ 的异常表达可导致包括乳腺癌在内的癌症的发生。迄今为止，大量证据表明这些异构体可以差异调节靶基因并调节其表达。因此，对单个异构体的定量分析可能有助于将临床结果与 p53 状态联系起来，并改善癌症患者的治疗效果。然而，关于 p53 异构体的准确测定的研究较少，这可能是由于这些异构体的序列同源性以及它们的低丰度所致。在这项研究中，开发了一种结合分子印迹聚合物 (MIPs) 和液相色谱-串联质谱 (LC-MS/MS) 的靶向蛋白质组学测定法，用于同时定量 C 末端 p53 异构体。首先选择同种型特异性替代肽 (即，用于同种型 α 的 KPLDGEYFTLQIR (肽-α)、用于同种型 β 的 KPLDGEYFTLQDQTSFQK (肽-β)和用于同种型 γ 的 KPLDGEYFTLQMLLDLR (肽-γ)) 用于 MIPs 富集和质谱检测。这三种替代肽的共同序列 KPLDGEYFTLQ 被用作 MIPs 的单一模板。除了优化印迹条件和表征制备的 MIPs 外，还评估了 MIPs 对每种替代肽的结合亲和力和交叉反应性。结果，实现了 5 nM 的 LOQ，比没有 MIPs 的灵敏度高 15 倍以上。最后，对该测定法进行了验证，并应用于几种人乳腺癌细胞系 (即 MCF-10A 正常细胞、MCF-7 和 MDA-MB-231 癌细胞以及耐药 MCF-7/ADR 癌细胞) 中 C 末端 p53 异构体 α、β 和 γ 的同时定量分析。这项研究是首次采用单模板 MIPs 和交叉反应现象来选择同种型特异性替代肽并实现基于 LC-MS/MS 的靶向蛋白质组学中蛋白质异构体的同时定量分析。
