Simultaneous quantification of Cyt c interactions with HSP27 and Bcl-xL using molecularly imprinted polymers (MIPs)...

Cytochrome c (Cyt c) plays an important role in cell apoptosis. However, it could be functionally compromised by interaction with anti-apoptosis proteins, known as protein-protein interactions (PPIs). Among the proteins potentially interacting with Cyt c, both HSP27 and Bcl-xL serve as pivotal anti-apoptosis proteins. Because multiple PPIs, especially those involve the same protein, could affect each other, their simultaneous and quantitative detection is highly needed. In this study, a combined approach of molecularly imprinted polymers (MIPs) and LC-MS/MS-based targeted proteomics was developed for simultaneous quantification of Cyt c-HSP27 and Cyt c-Bcl-xL interactions. Surrogate peptides of Cyt c, HSP27 and Bcl-xL were first selected and used for the corresponding proteins quantification in targeted proteomics analysis. For MIPs, epitope approach was employed and a short peptide of Cyt c was selected as template for protein complexes recognition and enrichment. The characteristics of the synthesized MIPs including adsorption capacity, kinetics and efficiency were then evaluated. After validation, this combined assay was applied to quantitative analysis of total Cyt c including Cyt c in mitochondria and cytosol, total HSP27, total Bcl-xL and Cyt c-HSP27 and Cyt c-Bcl-xL protein complexes in breast cells. The result was also compared with that using Co-IP/Western Blotting. SIGNIFICANCE: Protein-protein interactions (PPIs) are essential for many cellular processes and the changes of PPIs are often associated with cellular dysfunction. More importantly, each protein typically has more than one interaction partner and multiple PPIs, especially those involve the same protein, could affect each other. The selectivity of these interactions determines the activities of proteins and further the developmental potential of the cell. Thus, simultaneous and quantitative detection of multiple PPIs is highly needed in biological research and related disciplines. However, it is still challenging to even qualitatively or semi-quantitatively analyze multiple PPIs because of the limitations of current experimental techniques for interaction detection. In this study, molecularly imprinted polymers (MIPs) epitope approach was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) targeted proteomics for the simultaneous and quantitative detection of Cyt c-HSP27 and Cyt c-Bcl-xL interactions in breast cancer. Given high sensitivity, high selectivity and wide dynamic range of LC-MS/MS, MIPs approach was employed here to separate and enrich protein complexes prior to targeted proteomics analysis.