Blood-arachnoid barrier (BAB) is formed by the tight-junctioned arachnoid epithelial cells and acts as a blood-cerebrospinal fluid (CSF) interface. It has long been believed that the BAB is impermeable to water-soluble substances and plays a largely passive role. However, we recently demonstrated that the BAB is an active interface by in vivo studies for Slc22a6/Oat1, Slc22a8/Oat3 and Slco1a4/Oatp1a4 as model transporters. Our absolute quantitative proteomic analysis revealed that not only these transporters but also various transporters (ABCB1/P-gp, ABCG2/BCRP, SLC47A1/MATE1, SLC22A2/OCT2, SLC15A2/PEPT2, etc) are expressed at the BAB more abundantly than the blood-CSF barrier, although organic cation transporters (SLC47A1/MATE1 and SLC22A2/OCT2) were not detected in the mRNA expression analysis previously reported. It also revealed that the protein expression levels of transporters at the cerebral BAB are greater than those at the spinal cord BAB. The combination of plasma membrane separation and quantitative proteomics simultaneously clarified the plasma membrane localizations of multiple transporters in the arachnoid epithelial cells (CSF- or dura-facing plasma membrane) without any antibody. These results demonstrate that the quantitative proteomic techniques can overcome the issues in the mRNA expression and immunohistochemical analyses. Furthermore, these findings warn the idea that CSF drug concentrations are influenced by the blood-brain barrier transport system and the lumbar CSF concentrations are surrogates of brain interstitial fluid concentration. Here, on the basis of our findings obtained by proteomic studies, we introduce BAB transport system and discuss the physiological and pharmacological roles of the BAB.

The ribosome is a large RNA-protein complex, comprising four ribosomal RNAs and >80 ribosomal proteins. In human cells fully assembled ribosomes have a molecular weight of ~4.3 MDa and a diameter of 250–300 Å. During translation, multiple ribosomes can simultaneously engage the same mRNA to form ‘polysomes’. Centrifugation-based methods, particularly sucrose density gradients, have previously been used as the gold standard approach for separating polysomes from monosomes and free ribosome subunits. However, these gradient-based methods are technically complicated and time-consuming. We have developed an efficient new approach for mammalian polysome fractionation, called Ribo Mega-SEC. This separates polysomes and free ribosomal subunits by uHPLC, using Size Exclusion Chromatography (SEC) columns with a pore size of 2,000 Å. Using extracts from either cells, or tissues, polysomes can be separated, with high reproducibility, within 15 min from sample injection to fraction collection. Ribo Mega-SEC is readily combined with downstream analyses, including either electron microscopy, high-throughput MS-based proteomics, or RNA-Seq. The efficiency and reproducibility of the Ribo Mega-SEC method therefore facilitates a wide range of biochemical studies on polysomes and translation complexes in mammalian cells and tissues.

In this review, we would like to overview the recent trends of utilizing the single-molecular analysis methodologies to liquid biopsy. By establishing the system to detect single-molecules of biomolecules in less-invasively available clinical samples (e.g. blood and urine), the methodology has shown a potential to offer the excellent detection sensitivity and throughput, as exemplified by the development of next-generation DNA sequencers, which take advantage of multiplicity of single-molecule analysis to realize the significantly increased throughput. We will introduce the recent progresses in the field, mainly focusing on the technical advances in single-molecular protein analysis.