Comparison between LC-MS/MS and CE-LIF as a quantitative analysis methods for phosphorylated p53 peptides

Abstract

Post transcriptionally modified, tumor suppressor p53 protein plays a critical role in the regulation of genes that control cell cycle arrest, apoptosis and senescence. Therefore, finding a quantitative analyses of post-translational modifications are useful for predicting PTMs, biomarker discovery and understanding the relationship between protein modification and cancer development. We have developed quantitative capillary electrophoresis-laser-induced fluorescence (CE-LIF) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods for the analysis of phosphorylated p53 peptides. The synthetic substrate of p53 and its 4 phosphorylated products were used to validate the capabi- lity of the method. The substrate Pep1, which reached the detection window after 2.00 ± 0.02 min, and follow-ed by the phosphorylation products Pep2, Pep3, Pep4, and Pep5, which migrated slower. The LOD and LOQ obtained from CE-LIF and LC-MS/MS were within 11 ± 3.4 ng/mL and 33 ± 10 ng/mL, 0.02 ± 0.01 ng/mL and 0.07 ± 0.06 ng/mL, respectively. Both methods showed similar reproducibility and accuracy. The reproducibility and accuracy was within 9.2 % and 105%, respectively. Because of its low limit of detection and accurate quantitative analysis ability, both methods have the potential for high throughput screening of phosphorylated p53 protein for discovery of therapeutic target and drug program.