Development of a UPLC-MRM-based targeted proteomic method to profile subcellular organelle marker proteins from human liver tissues

Abstract

AbstractSubcellular organelles have long been an interest in biochemical research and drug development as the isolation of those organelles can help to probe protein functions and elucidate drug disposition within the cell. Usually, the purity of isolated subcellular organelle fractions was determined using immunoblot analysis of subcellular organelle marker proteins, which can be labor-intensive and lack reproducibility due to antibody batch-to-batch variability. As such, a higher throughput and more robust method is needed. Here, a UPLC-MRM-based targeted proteomic method was developed for a panel of human organelle marker proteins and used to profile a series of sucrose fractions isolated from the protein extract of human liver tissues. The method was validated by comparing to the traditional immunoblot and determining subcellular localization of three case study proteins (CYP3A4, FcRn, and β2M) pertaining to the disposition of small molecule and biologic drugs. All three case study proteins were co-enriched with their corresponding subcellular protein marker, and complete recoveries were achieved from isolated fractions. This newly developed MRM method for the panel of human organelle marker proteins can potentially accelerate future intracellular drug disposition analysis and facilitate subcellular organelle quality assessment.