Background

The identification of unique senescence markers remains challenging. Current hallmarks of senescent cells, including increased senescence-associated β-galactosidase activity, increased levels of cell cycle regulators such as p16^INK4a, p27, and p53, and altered levels of sirtuins and lamins, are detected commonly by Western blot and immunohistochemistry methods. Mass spectrometry outperforms these conventional quantification methods in terms of high throughput, specificity, and reproducibility.

Objectives

To develop multiple reaction monitoring-based tandem mass spectrometric senescence assay for simultaneous measuring of p16^INK4a, p27, p53, p53-β, the seven proteins of the sirtuins family and the four transcript variants of lamins proteins in aging cell model and cancerous cell lines.

Methodology

Multiple reaction monitoring-tandem mass transitions per protein were developed for each signature peptide(s) and stable isotope-labeled internal standard. The developed assay was validated in a matrix using breast cancer MCF7 cell lines according to the US-FDA guidelines for bioanalytical assays.

Results

The analytes chromatographic peaks were baseline separated and showed linear behavior in a wide dynamic range with r² ≥ 0.98. The method for all proteins has passed the inter/intra-day precision and accuracy validation using three levels of quality control samples. The accuracy and the precision for most analytes were 80–120% and ≤20%, respectively. The method's sensitivity for the panels' signature peptides ranged from 1 ng μL⁻¹ to 1 μg mL⁻¹. Extraction recovery assessed in two quality control levels was >60% for most analytes. This LC-MS-MS validated senescence assay showed reduced lamin A, lamin A△10, lamin A△50, SIRT1, SIRT3, SIRT5, p53, and p16^INK4a, as well as p53-β induction, are implicated in replicative senescence. Meanwhile, increased lamin C: lamin A ratio was evident and can diagnose breast carcinogenesis. Moreover, in breast cancer metastasis, reduced SIRT2 and p27 and elevated levels of lamin A△50, SIRT5, SIRT7, and p53-β are evident.

Conclusion

LC-MS/MS is a potent alternative tool to the currently available assays. The high throughput method established can study senescence's role in different pathophysiological processes.

中文翻译：

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使用多反应监测液相色谱-串联质谱法对细胞中的衰老介质进行绝对定量

背景

独特的衰老标志物的鉴定仍然具有挑战性。衰老细胞的当前特征，包括衰老相关的 β-半乳糖苷酶活性增加，细胞周期调节因子（如 p16 ^INK4a、p27 和 p53）水平增加，以及沉默调节蛋白和核纤层蛋白水平改变，通常通过蛋白质印迹和免疫组织化学方法检测到。质谱在高通量、特异性和重现性方面优于这些传统的定量方法。

目标

开发基于多反应监测的串联质谱衰老测定法，用于同时测量衰老细胞模型和癌细胞系中的 p16 ^INK4a、p27、p53、p53-β、sirtuins 家族的七种蛋白质和 lamins 蛋白质的四种转录变体.

方法

针对每个特征肽和稳定同位素标记的内标，开发了每种蛋白质的多重反应监测串联质量转换。根据美国 FDA 的生物分析检测指南，使用乳腺癌 MCF7 细胞系在基质中验证了开发的检测方法。

结果

分析物色谱峰是基线分离的，在r ² ≥ 0.98的宽动态范围内显示线性行为。所有蛋白质的方法都通过了使用三级质量控制样品的日间/日内精密度和准确度验证。大多数分析物的准确度和精密度分别为 80-120% 和 ≤20%。该方法对面板特征肽的灵敏度范围从1 ng μL ^-1到1 μg mL ^-1。对于大多数分析物，在两个质量控制水平上评估的提取回收率 >60%。该 LC-MS-MS 验证的衰老测定显示 lamin A、lamin A△10、lamin A△50、SIRT1、SIRT3、SIRT5、p53 和 p16 ^{INK4a 减少}以及 p53-β 诱导与复制衰老有关。同时，核纤层蛋白C：核纤层蛋白A比值升高是明显的，可以诊断乳腺癌的发生。此外，在乳腺癌转移中，SIRT2 和 p27 降低以及 lamin A△50、SIRT5、SIRT7 和 p53-β 水平升高是明显的。

结论

LC-MS/MS 是当前可用分析的有效替代工具。建立的高通量方法可以研究衰老在不同病理生理过程中的作用。