Extracellular vesicles (EVs) are cell-derived microparticles present in most body fluids, mainly including microvesicles and exosomes. EV-harbored proteins have emerged as novel biomarkers for the diagnosis and prediction of different cancers. We successfully isolated microvesicles and exosomes from human saliva, which were further characterized comprehensively. Salivary EV protein profiling in normal subjects and lung cancer patients was systematically compared through utilizing LC-MS/MS-based label-free quantification. 785 and 910 proteins were identified from salivary exosomes and microvesicles, respectively. According to statistical analysis, 150 and 243 proteins were revealed as dysregulated candidates in exosomes and microvesicles for lung cancer. Among them, 25 and 40 proteins originally from distal organ cells were found in the salivary exosomes and microvesicles of lung cancer patients. In particular, 5 out of 25 and 9 out of 40 are lung-related proteins. Six potential candidates were selected for verification by Western blot, and four of them, namely, BPIFA1, CRNN, MUC5B, and IQGAP, were confirmed either in salivary microvesicles or in exosomes. Our data collectively demonstrate that salivary EVs harbor informative proteins that might be used for the detection of lung cancer through a noninvasive way.

We found that highly metastatic lung cancer cell derived sEVs can regulate the phenotype of recipient cells. With the help of quantitative proteomics, cancer cell metastasis related sEVs proteins were revealed. Through in vitro and in vivo experiments, we found that sEVs-HGF can promote cancer metastasis through activating c-Met pathway. This finding provides insights into how cancer cells send carcinogenic information to remote cells and regulate their fate, which might in turn be targeted in cancer therapy.

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Bombesin-like receptor 3 (BRS3), an orphan G protein-coupled receptor (GPCR), plays important roles in our biological system while the exact mechanisms behind it are less known. To get insights of the biological effects upon BRS3 activation, we utilized quantitative proteomics approach to explore the dynamic protein profiling during the stimulation by its ligand. At different time points after stimulation with BRS3 surrogate agonist, the protein profiling in BRS3 overexpressed HEK 293 cells BRS3 (HEK 293-BRS3) was analyzed by nano-LC–MS/MS. In total, 1593 cellular proteins were confidently identified and quantified, including 146 proteins dysregulated at multiple time points and 319 proteins only altered at one time point. Data analysis indicated that BRS3 activation could regulate cell death, survival, and protein synthesis, particularly mRNA translation. Key signaling pathways were revealed for BRS3 signal transduction. In particular, 21 of our identified proteins are involved in the rapamycin (mTOR) signaling pathway. The promotion of mTOR was further confirmed through monitoring its indicative targets upon BRS3 activation. Upon the inhibition of mTOR by rapamycin, cell proliferation was dramatically reversed. Our proteomics data collectively demonstrate that BRS3 activation will lead to cascades of signal transduction and promote cell proliferation. The developed strategy might be utilized to discover the roles of other GPCRs and improve our understanding of their unknown functions.

Human microbiome contains billions of microorganisms that play important roles in the biological system and different diseases. Due to its complexity, conventional culture-independent technology may underestimate the value of low-abundance bacteria, which calls for a highly efficient method for its enrichment and comprehensive analysis. In this study, we developed a recycling free-flow isoelectric focusing (RFFIEF) method-based electrophoresis method to separate salivary microbiome. First, we used Escherichia coli (DH5α) as a model for RFFIEF method development, which was focused in a narrow pH range (0.38 pH unit). The recovery rate was 80.81% with 5.85% relative standard deviation (n = 5). The optimized method was then adopted to separate the human salivary microbiome into 32 fractions, followed by 16S rRNA gene sequencing and metaproteomics analysis. After RFFIEF fractionation, we identified 508 bacterial genera, which increased by 225% on average (n = 3) when compared to the results before fractionation. We further compared the compositional change of microbiome in the saliva of lung cancer group (n = 22) and control group (n = 21) through RFFIEF. Quantitative results demonstrated that six bacterial genera were upregulated dramatically in the lung cancer group, while two genera were downregulated. Through qPCR verification in an independent sample set (n = 48), we confirmed that genus Granulicatella was significantly upregulated in the lung cancer group, whereas Pseudomonas was remarkably downregulated (p < 0.001). RFFIEF is an efficient and reproducible technology to fractionate the microbiome for its comprehensive analysis, which can be further applied to the in-depth study of the complex microbiomes and contribute to the discovery of disease-associated bacteria.