Comparative Proteomic Analysis of Exosomes and Microvesicles in

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Comparative Proteomic Analysis of Exosomes and Microvesicles in Human Saliva for Lung Cancer Yan Sun, Chunhui Huo, Zhi Qiao, Zhi Shang, Asad Uzzaman, Sha Liu, Xiaoteng Jiang, Liu-Yin Fan, Liyun Ji, Xin Guan, Cheng-Xi Cao, and Hua Xiao J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00770 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Proteome Research

Comparative Proteomic Analysis of Exosomes and Microvesicles in Human Saliva for Lung Cancer

Yan Sun a, Chunhui Huo a, Zhi Qiao a, Zhi Shang a, Asad Uzzaman a, Sha Liu a, Xiaoteng Jiang a, Liu-Yin Fan a, Liyun Ji a, Xin Guan b,*, Cheng-Xi Cao a,*, and Hua Xiao a,*

a

State Key Laboratory of Microbial Metabolism, School of Life Sciences and

Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China b

Department of Thoracic Surgery, Ninth People's Hospital, School of Medicine,

Shanghai Jiao Tong University, Shanghai, 200011, China

* Corresponding Authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China. Tel: +86-21-3420-5682 (H. Xiao) Email addresses: [email protected] (H. Xiao); [email protected] (C.X. Cao); [email protected] (X. Guan) 1

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Abstract: Extracellular vesicles (EVs) are cell-derived microparticles present in most body fluids, mainly including microvesicles and exosomes. EVs-harbored proteins have emerged as novel biomarkers for the diagnosis and prediction of different cancers. Herein, we successfully isolated microvesicles and exosomes from human saliva, which were further characterized comprehensively. Salivary EVs 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 that 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. 6 potential candidates were selected for verification by western blot, and 4 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 non-invasive way.

Keywords: extracellular vesicles; saliva; exosomes; microvesicles; proteomics; lung cancer 2


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Introduction Extracellular vesicles (EVs) are microparticles released by a variety of cells into the extracellular microenvironment. EVs could selectively packing different cargos, such as nucleic acids and proteins, and transport them into the acceptor cells, leading to changes in resident cells and modulation of cell fates[1-2]. Recent studies suggest that EVs in body fluids could serve as non-invasive biomarkers and have attracted intensive interests in basic and translational cancer research[3]. Oncogenic information carried by EVs could create an environment that favors tumor growth, metastasis, and cancer progression[4]. Therefore, comprehensive analysis of EVs components could lead to finding of novel candidates for cancer detection and therapy. Human saliva has been increasingly used as a biofluid for both research and clinical applications. Since saliva can be collected through non-invasive manner and processed easily, salivary diagnostics would contribute to personalized medicine through delivering patient-centric healthcare[5-8]. Many studies have attempted to use salivary EVs for biomarker discovery[9-11], including microvesicles and exosomes, the most common components of EVs. Microvesicles are originated directly from the plasma membrane, and are often classified as ectosomes, which ranged from 100-1000 nm[12]. Meanwhile, exosomes are generally thought to derive from the endosomal compartment with the size of 30-100 nm[13]. Unraveling the proteomic of microvesicles and exosomes is of biological importance that might shed light on extracellular 3



communication and result in clinical diagnosis of cancer. The goal of the current study was to isolate microvesicles and exosomes from human saliva and in depth characterize their protein profiling. The potential of EVs as biomarker source for the detection of lung cancer is discussed.

Materials and methods Saliva collection and preparation Saliva samples were collected according to the approved protocols (IRB#M15017) by Bio-X Ethics Committee of Shanghai Jiao Tong University and all subjects provided written informed consents. Briefly, unstimulated saliva samples were kept on ice during the sample collection, which supplemented with EDTA-free Protease Inhibitor Cocktail (Roche Applied Science, Basel, Switzerland). The samples were centrifuged at 2600 g for 30 min at 4 ℃ to remove insoluble materials and cell debris and stored at -80 ℃ for further use. Microvesicles and exosomes isolation Prior to EVs isolation, saliva samples were passed through affinity chromatography column combined with filter system (ACCF) to remove high abundant proteins, as previously described[14]. Then microvesicles and exosomes were isolated through the experimental workflow as shown in Figure 1. Briefly, saliva samples were centrifuged at 10,000 or 20,000 ×g for 1 h at 4 ℃ to separate the salivary microvesicles. The supernatant was further ultracentrifugated at 100,000 or 125,000 g for 2.5 h at 4 ℃ to pellet the exosomes. 4


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SDS-PAGE Salivary microvesicles and exosomes proteins were extracted and then loaded into a 10% Bis-Tris Mini Gel (InvitrogenTM ThermoFisher). The SDS-PAGE was run at 120 V for 90 mins in MOPS running buffer. The resulting gels were stained with Fast Sliver Stain Kit (Beyotime, Shanghai, China). Nanoparticle tracking analysis (NTA) Nano-Sight LM10 instrument (Malvern, London, UK) was used to analyze the size distribution of salivary microvesicles and exosomes. The diluted samples were illuminated by a monochromatic laser beam at 488 nm to register a 60 second video taken with a mean frame rate of 30 frames per second. Bio-transmission electron microscopy Transmission electron microscopic analysis was performed for isolated vesicle fractions as follows. 10 µL of each sample was placed on Formvar/carbon coated TEM grids (AGH300; Zhong Jing Ke Yi Technology) for 10 mins at room temperature. The grids were removed, blotted with filter paper and placed onto drops of freshly prepared 5% phosphotungstic acid for 2 mins. The excess solution was removed and the grids were air-dried. The images of microvesicles and exosomes were captured using the Philips CM120 transmission electron microscopes (Tecnai G2 spirit Biotwin, FEI, USA). Nano LC-MS/MS analysis and Label-free quantification Filter aided proteome preparation(FASP) tryptic digestion was carried out overnight at 37 ℃ using trypsin (Promega, Madison, WI, USA). 1 µg protein 5



digests from each sample were resuspended and analyzed by nanoLC-MS/MS (Thermo Scientific Q Exactive, USA). For protein identification, spectra were searched against the Human SwissProt database (548208 sequences). All searches were performed with tryptic specificity allowing up to two missed cleavages. Oxidation and acetylation of protein N termini were considered as variable modifications. The mass tolerance values for precursor and fragment ions were 6 ppm and 0.5 Da, respectively. The criteria of 2 unique peptides, p

Comparative Proteomic Analysis of Exosomes and Microvesicles in

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