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

Subcellular 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 ß2M) 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.

Minimal Cerebrospinal Fluid Concentration of Miltefosine despite Therapeutic Plasma Levels during the Treatment of Amebic Encephalitis.

Miltefosine is an alkylphosphocholine compound that is used primarily for treatment of leishmaniasis and demonstrates in vitro and in vivo antiamebic activity against Acanthamoeba species. Recommendations for treatment of amebic encephalitis generally include miltefosine therapy. Data indicate that treatment with an amebicidal concentration of at least 16 µg/ml of miltefosine is required for most Acanthamoeba species. Although there is a high level of mortality associated with amebic encephalitis, a paucity of data regarding miltefosine levels in plasma and cerebrospinal fluid in vivo exists in the literature. We found that despite aggressive dosing (oral miltefosine 50 mg every 6 h) and therapeutic plasma levels, the miltefosine concentration in cerebrospinal fluid was negligible in a patient with AIDS and Acanthamoeba encephalitis.

Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis.

The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality.