Exosomes are secreted nanovesicles shed by almost all kinds of cells. Recently, increased interest has been focused on these extracellular vesicles as natural carriers transporting biological contents for intercellular communication. However, current isolation techniques, such as ultracentrifugation, are not convenient and often require specialized equipment. Herein, we describe a polyethylene glycol (PEG)-based approach, which could permit facile, low-cost and effective isolation of exosomes from cell culture supernatant. High-resolution electron microscopes clearly visualized the size and morphology of isolated exosome aggregates, implying the mechanism of PEG-based precipitation. Combined with tandem mass spectrometry analysis, 6299 protein groups encoded by 5120 genes were successfully characterized from HeLa cell culture supernatant, including numerous exosome proteins which could overlap 97% of the Top 100 exosome marker proteins recorded in the ExoCarta database, as well as a series of low-abundance cytokines and biomarkers. Furthermore, we found a higher ratio of neo-cleavage sites in proteins identified from exosomes compared with cellular proteins, revealing the potential roles of exosomes in accumulation and transportation of protein degradation intermediates.

Human epithelial cell culture models of monolayer Caco-2 cells have been widely employed to assess the absorption of drug molecules across intestinal mucosa. However, cautions should be taken when interpreting the conclusions from those models due to their undesirable phenotype and functionality when compared with the native intestinal tissue. In the present study, an improved, more physiologically relevant three-dimensional (3D) culture model of the intestinal mucosa was developed to study drug absorption, in which a coculture of epithelial cells, including Caco-2 cells and HT29-methotrexate cells, was indirectly seeded on a Transwell filter insert with collagen gel and stromal cells (fibroblasts and immunocytes) incorporation. This setting-up provided a compatible environment to improve the phenotype and functionality of the epithelial cells. Compared with the monolayer culture of Caco-2 cells, the reconstructed 3D model displayed more physiologically relevant characteristics evidenced by its decreased TEER value and mucus-like layer formation. A decreased expression of P-gp and an increased expression of BCRP were also observed in the current 3D culture model, leading to a changed secretory permeability of their substrates. More importantly, an improved correlation (R(2)=0.843) was obtained between the absorptive permeability across the 3D coculture model and the human absorption fraction especially for those model compounds with moderate or high permeability. Thus, this engineered 3D coculture model presents a unique, improved opportunity to evaluate drug permeability in vitro.

No abstract

The analysis of protein N-termini is of great importance for understanding the protein function and elucidating the proteolytic processing. Herein, we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted N-termini enrichment (HYTANE) to achieve a global N-terminome analysis. The HYTANE strategy showed a high efficiency in hydrophobic tagging and C18 material-assisted depletion using bovine serum albumin (BSA) as the sample. This strategy was applied to N-termini profiling from S. cerevisiae cell lysates and enabled the identification of 1096 protein N-termini, representing the largest N-terminome data set of S. cerevisiae. The identified N-terminal peptides accounted for 99% of all identified peptides, and no deficiency in acidic, histidine (His)-containing, and His-free N-terminal peptides was observed. The presented HYTANE strategy is therefore a highly selective, efficient, and unbiased strategy for the large scale N-terminome analysis. Furthermore, using the HYTANE strategy, we identified 329 cleavage sites and 291 substrates of caspases in Jurkat cells, demonstrating the great promise of HYTANE strategy for protease research. Data are available via ProteomeXchange with identifier PXD004690.

No abstract

A novel kind of aptamer modified organic-inorganic hybrid silica monolithic capillary column has been developed, via the covalent bonding of 5'-NH(2)-modified aptamer for human α-thrombin on hybrid silica monolith, prepared by sol-gel method, with tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors. Due to the large specific surface area of the hybrid matrix, the average coverage density of aptamer reached 568 pmol/μL, and the thrombin binding capacity was 1.15 μg/μL, 14 times higher than that of aptamer modified open tubular capillaries. By such an affinity capillary column, the limit of detection of thrombin was decreased to 3.4 nM with a UV detector. Furthermore, even when thrombin was mixed with 1000 times more concentrated human serum, it could be selectively enriched and detected with the signal-to-noise ratio as ca.10. These results indicate that the developed preparation strategy for aptamer based hybrid silica monolithic capillary column might provide an effective method to achieve highly selective recognition of trace targets.

Pancreatic cancer is a highly metastatic and chemo-resistant disease. Secreted proteins involved in cell-cell interactions play an important role in changing the tumor microenvironment. Previous studies generally focus on the secretome of cancer cell line from serum-free media, due to the serious interference of fetal bovine serum (FBS). However, serum-starvation may alter expression patterns of secreted proteins. Hence, efforts to decrease the interference of serum in proteomic analysis of serum-containing media have been hampered to quantitatively measure the tumor secretion levels. Recently, the metabolic labeling, protein equalization, protein fractionation and filter-aided sample preparation (FASP) strategy (MLEFF) has been successfully used to avoid the disturbance of serum on secretome analysis. Here, this efficient method was applied for comparative secretome analysis of two hamster pancreatic cancer cells with differentially metastatic potentials, enabling the observation of 161 differentially expressed proteins, including 106 proteins that had been previously reported and detected in plasma. By integrated analysis of our data and publicly available bioinformatics resources, we found that a combination panel consisting of CDH3, PLAU, and LFNG might improve the prognosis of overall pancreatic cancer survival. These secreted proteins may serve as a potential therapeutic targets for pancreatic cancer metastasis.

Secreted proteins play key roles during cellular communication, proliferation, and migration. The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been performed under serum-free conditions. However, these conditions might alter the status of the cells. Herein, we describe an efficient strategy that avoids the disturbance of serum by combining metabolic labeling, protein "equalization," protein fractionation, and filter-aided sample preparation, called MLEFF, enabling the identification of 534 secreted proteins from HeLa conditioned media, including 31 cytokines, and growth factors. This MLEFF strategy was also successfully applied during a comparative secretome analysis of two human hepatocellular carcinoma cell lines with differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumor invasion and metastasis.