Absolute quantification of senescence mediators in cells using multiple reaction monitoring liquid chromatography-Tandem mass spectrometry

Abstract

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.

Keywords: Bottom-up approach; Cell cycle regulators; Lamins; Multiple reaction monitoring, and liquid chromatography-tandem mass spectrometry; Senescence; Senescence biomarkers; Sirtuins.