LC-MS/MS-based Targeted Proteomics Quantitatively Detects The Interaction Between P53 And MDM2 In Breast Cancer

In breast cancer,p53 could be functionally compromised by interaction with several proteins.Among those proteins,MDM2 serves as a pivotal negative regulator and counteracts p53 activation.Thus,the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction(PPI),provide a better insight into cancer development and allow the emergence of advanced treatments.However,rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far.The major issue of current available approaches is the trade-off between sensitivity and specificity.Thus,techniques with the ability to quantify PPIs with both high sensitivity(low false-negative rate)and high specificity(low false-positive rate)are eagerly desired.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)-based targeted proteomics has shown the potential to study biomolecules because of its high sensitivity,high selectivity and wide dynamic range.In this study,a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation(Co-IP)for the quantification of p53-MDM2 complex.A p53 antibody with the epitope residing at 156-214 residues achieved the greatest IP efficiency.321KPLDGEYFTLQIR333(p53)and 327ENWLPEDK334(MDM2)were selected as surrogate peptides in the targeted analysis.Stable isotope-labeled synthetic peptides were used as internal standards.An LOQ(limit of quantification)of 2 ng/mL was obtained.Then,the assay was applied to quantitatively detect total p53,total MDM2 and p53-MDM2 in breast cells and tissue samples.Western blotting was performed for a comparison.Finally,a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored.