Quantitiative Ce-LIF and LC-MS/MS methods for phosphorylated p53 peptides

Abstract

The tumor suppressor p53 protein plays an important role in regulating genes that control cell cycle arrest, apoptosis, and cell senescence. The phosphorylation of p53 protein remains low in normal cells and is activated via PTMs when cells encounter stressors. Therefore, finding a quantitative analysis method of PTM is useful for understanding the relationship between protein modification site, interacting enzymes which lead to cell senescence and cancer incidence. In this study, capillary electrophoresis-laser induced fluorescence (CE-LIF) and liquid chromatography-LTQ-Orbitrap mass spectrometry (LC-MS/MS) methods were developed for the quantitative analysis of phosphorylation p53 peptide at n-terminal. The method was validated using the synthetic substrate of p53 and its four phosphorylated peptides at 6, 9, 15, and 20 serine. The LOD and LOQ obtained from CE-LIF were as follows; 0.2 and 0.6 ng/mL (Pep1), 1.6 and 4.8 ng/mL (Pep2), 0.4 and 1.2 ng/mL (Pep3), 0.6 and 1.8 ng/mL (Pep4), 1.2 and 3.6 ng/mL (Pep5). Compared to CE-LIF method, LC-MS/MS method showed higher LOQ values; 27 ng/mL, 12 ng/mL, 18 ng/mL, 9 ng/mL, and 24 ng/mL, from Pep1 to Pep5 respectively. The reproducibility and accuracy of QC samples were within 9.5% and 94 ± 5.1% for CE-LIF and 12% and 99 ± 8.5% for LC-MS/MS, respectively. The developed methods were applied to quantitative analysis of target peptides in colon cancer cell line, HCT116, treated with different concentrations of doxorubicin. The results showed that decrease of non-phosphorylated p53 peptide and elevation of phosphorylated p53 peptides. Both methods gave low detection limit and wide quantitative ranges. However CE-LIF method surpasses LC-MS/MS method with its lower detection limit and less sample amount requirement. Therefore, CE-LIF has the potential for high throughput screening of phosphorylated p53 protein for discovery of therapeutic target and drug screening for anti-cancer therapy.