How Long Can Be Exosomes Be Out of Fridge Before It Goes Bad
J Extracell Vesicles. 2017; 6(1): 1359478.
Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses
Rosario Maroto
a Sealy Center for Molecular Medicine, University of Texas Medical Branch (UTMB), Galveston, TX, The states
b Institute for Translational Sciences, UTMB, Galveston, TX, Usa
Yingxin Zhao
a Sealy Center for Molecular Medicine, University of Texas Medical Branch (UTMB), Galveston, TX, USA
b Establish for Translational Sciences, UTMB, Galveston, TX, U.s.a.
c Department of Internal Medicine, UTMB, Galveston, TX, USA
Mohammad Jamaluddin
b Plant for Translational Sciences, UTMB, Galveston, TX, Usa
c Department of Internal Medicine, UTMB, Galveston, TX, The states
Vsevolod 50. Popov
d Department of Pathology, UTMB, Galveston, TX, USA
Hongwang Wang
e Department of Chemistry, Kansas State University, Manhattan, KS, USA
Madumali Kalubowilage
due east Section of Chemistry, Kansas State University, Manhattan, KS, USA
Yueqing Zhang
c Department of Internal Medicine, UTMB, Galveston, TX, The states
Jonathan Luisi
f Centre for Biomedical Engineering, UTMB, Galveston, TX, USA
Hong Dominicus
c Section of Internal Medicine, UTMB, Galveston, TX, United states of america
Christopher T. Culbertson
e Department of Chemistry, Kansas State University, Manhattan, KS, USA
Stefan H. Bossmann
e Section of Chemistry, Kansas State Academy, Manhattan, KS, USA
Massoud Motamedi
f Eye for Biomedical Applied science, UTMB, Galveston, TX, Usa
Allan R. Brasier
a Sealy Center for Molecular Medicine, Academy of Texas Medical Co-operative (UTMB), Galveston, TX, U.s.
b Found for Translational Sciences, UTMB, Galveston, TX, USA
c Section of Internal Medicine, UTMB, Galveston, TX, United states of america
Received 2022 December 22; Accepted 2022 Jul sixteen.
ABSTRACT
Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions demand to accept minimal touch on airway exosome integrity.
Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing.
Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −fourscore°C. Exosomal structure was assessed by dynamic low-cal scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified past label-complimentary LC-MS/MS.
Results: Freshly isolated BALF exosomes exhibited a mean bore of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a ten% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −lxxx°C produced an even greater effect, resulting in a 25% increment in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +iv°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins correspond exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at −lxxx°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures.
Conclusions: Storage destabilizes the surface characteristics, morphological features and poly peptide content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately subsequently isolation.
KEYWORDS: Extracellular vesicles, bronchoalveolar lavage, storage conditions, label-complimentary, quantitative proteomics
Introduction
Exosomes are small <150 nm extracellular vesicles derived from endosomal multivesicular bodies, and part in extracellular indicate transduction.[ane,ii] Enclosed inside protective phospholipid bilayers, exosomes contain proteins, vasoactive leukotrienes, and small-scale RNAs whose composition dynamically changes with the cellular microenvironment.[ii] We currently empathize that exosomes bear on cellular behaviour through paracrine actions on bespeak transduction pathways in distal cells. With a greater understanding of how the exosome content is afflicted by cellular responses to its microenvironment, profiling and quantification of the exosome content may exist used every bit "liquid biopsies" to discover occult cellular stress and inflammation.[iii] Although much study has focused on exosomes in the circulation, all cell types produce exosomes, which consequently are constitute in nigh every biological fluid, including the airways.
Asthma is a chronic inflammatory lung disease that affects over 300 million adults and children, making information technology a major public health problem.[4,5] In asthma, epithelial damage and repair is a major pathophysiological procedure mediating the progression.[6] Although epithelial injury in asthma is exacerbated past aero-allergens and ecology pollutants, recurrent airway infections are the most common cause of episodic decompensation.[seven] Over time, these exacerbations lead to an accelerated turn down in lung part, bookkeeping for significant morbidity and bloodshed.[8,ix] Understanding how viruses trigger epithelial inflammation and remodelling is therefore a clinically important question.
The airway mucosa senses viruses and initiates the inflammatory response by detecting molecular patterns unique to viral replication.[10,11] One of these, extracellular dsRNA [poly(I:C)], is a potent viral molecular pattern that triggers innate inflammation through the toll-similar receptor (TLR)-3 pathway, resulting in rapid recruitment of neutrophils.[12,13] Genomic and proteomic studies have shown that viruses trigger airway epithelial cells to limited and secrete over 570 proteins including soluble type I and type III interferons (IFNs),[xiv–16] and C-, CXC- and CC-type chemokines.[17–21] These proteins participate in inducing antiviral protection and activate adaptive amnesty.[eighteen–21] Our recent systematic study discovered that exosome secretion was a pregnant component of the epithelial innate anti-viral response, bookkeeping for approximately 1/3 of all the secreted proteins.[18] Although we know that exosomes constitute a pregnant component of the virus-induced innate response, their functional office in the exacerbations and progression of asthma is poorly understood.
A better insight into how airway inflammation changes exosome content will advance our understanding of their functional office in airway remodelling and advance the evolution of sensitive preclinical assays for regional remodelling. Sampling the bronchoalveolar lavage fluid (BALF) provides a ways to selectively study the condition of the proximal airway mucosa.[22] Studies examining BALF exosomes isolated from atopic asthmatics accept shown that these exosomes have differential cytokine, leukotriene and miRNA content than normal; these biomolecules could potentially regulate bronchial hyperresponsiveness and inflammation.[23,24] Exosomes isolated from ciliated tracheal epithelial cells mediate a protective innate allowed response past the expression of sialoproteins that block flu infection.[25] Others have found that IL-13, a mediator of allergy and Th2 lymphocyte recruitment in asthma, induces exosome production in the airway, promoting chemotaxis of macrophages.[26] Understanding the biological processes mediated by exosomes will provide major new insights into respiratory disease.
For unbiased analysis of exosome function, standard methods should exist identified that maintain exosomes in their original biological state every bit much as possible. Although exosomes are widely thought to be biologically inert vesicles that can be stored for long periods of fourth dimension,[27] and are stable upon freezing,[28] nosotros observed that the biophysical and protein content of airway exosomes dramatically change upon storage. We therefore conducted a systematic concrete characterization and proteomic analysis of BALF exosomes under different temperatures. Our data bespeak that storage temperature affects BALF exosome morphology, size and protein content, and this behaviour should exist taken into consideration in any diagnostic or mechanistic studies.
Materials and methods
Materials
Polyinosinic–polycytidylic acid poly (I:C) was obtained from Sigma (St Louis, MO, Us) as the sodium salt and freshly prepared in phosphate buffered saline (PBS; 300 μg/40 µl). All PBS used in poly (I:C) administration and exosome isolation was Ca++ and Mg++-free. All reagents and solvents in LC-MS/MS analyses were ACS grade. Ammonium bicarbonate (ABC),2,two,ii,-trifluoroethanol(TFE), and acetic acid were purchased from Sigma-Aldrich. Iodoacetamide (IDA), dithiothreitol (DTT), acetonitrile (ACN), formic acid, and methanol were purchased from Thermo Scientific (Waltham, MA, USA). Urea ultra was from MP Biomedicals (Santa Ana, CA, The states). Sequencing-course modified trypsin and LysC were from Promega (Madison, WI, USA).
Bronchoalveolar lavage (BAL) fluid drove
Male person mice (strain C57BL/6J, viii–x weeks sometime, 25–30 g) were purchased from Jackson Laboratory (Bar Harbor, ME, United states of america) and housed according to IACUC protocol# 1312058 (UTMB). For stimulation, mice were lightly anaesthetized and poly (I:C) solution was administered slowly via the intranasal route, alternating nostrils. BALF was collected 24 h later. Anaesthetized mice were tracheostomized and BALF collected by gentle lavage with 1 ml sterile PBS three times. The total BALF recovered (about 3.five ml) was kept at +4°C and immediately processed for exosome isolation.
Exosome isolation
Isolation of exosomes was performed by differential centrifugation of the BALF post-obit established centrifugation times and parameters.[29,30] All centrifugations and procedures were carried out at +iv°C to minimize poly peptide deposition. Cells nowadays in the BALF were removed by depression-speed centrifugation at 400 × 1000 for ten min. The cleared supernatant was then sequentially centrifuged at 2000 × g for 15 min (ThermoScientific IEC CL31R multispeed centrifuge, rotor T41*11210435) and ten,000 × one thousand for 30 min to remove any remaining cell debris/microvesicles (Beckman Optima TLX ultracentrifuge, rotor TLA-100.three, Indianapolis, IN, The states). Exosomes were finally pelleted past ultracentrifugation at 100,000 × yard for ii h and done in PBS at 100,000 × thou, 60 min. After washing, the exosomal pellet was carefully resuspended in a full of 300 µl PBS, divided into aliquots (100 µl each) and analysed immediately (fresh) or stored for four days at +4°C or −fourscore°C. Afterwards four days, both exosomal samples were placed on ice, allowing the −eighty°C sample to thaw. Exosomes in both aliquots were re-pelleted by an boosted centrifugation (100,000 × g for 2h) and supernatants collected. Exosomes were evaluated by size and morphological features using dynamic low-cal scattering and transmission electron microscopy. Supernatants were concentrated using Amicon ultra-4 centrifugal filters-3K (Millipore, Billerica, MA, U.s.). Both exosomes and supernatants were nerveless for LC-MS/MS analysis.
Dynamic light scattering (DLS)
A x µl aliquot from the resuspended exosome sample was diluted in 990 µl of PBS, mixed well and loaded into the cuvette. Three determinations per sample were taken at room temperature using a Malvern High Performance Particle Sizer (HPPS, incorporating non-Invasive Back Scatter Technologies, Malvern Instruments, Westborough, MA, The states) for each contained experiment. The exosome size was calculated using the Stokes–Einstein equation to determine the particle'due south hydrodynamic radius (Rh) or diameter. In cursory, the Brownian motion of a particle is measured past the fluctuations of scattered light intensity at a fixed angle (173°), laser wavelength 633 nm, as an indication of the velocity distribution of the particle movement in solution. Exosomes suspended in a sample volume of 1 ml of PBS was measured and a full of three readings per sample were performed. Information acquisition and assay were performed using Dispersion Technology Software (DTS, V4.1.26.0, Bedford Hills, NY, USA) configured for HPPs assay. DTS analysis allows 1 to interpret the data acquired considering several parameters, such equally intensity, book and number distribution, every bit well every bit statistical analysis. The average particle bore results from a top of a Gaussian model fitting to the particle distribution, and the polydispersity index (Pdl) reflects the width of the primary size distribution nowadays in the solution. To ensure proper operation of the musical instrument the equipment was calibrated periodically using polymer latex spheres (Malvern).
Transmission electron microscopy (TEM)
A 10 µl aliquot from the exosome interruption was diluted in deionized water, applied to 200 mesh Formvar/carbon coated copper grids (Electron Microscopy Sciences, Hatfiled, PA, U.s.a.) for x min at room temperature (24°C) and negatively stained with 2% uranyl acetate (UA). The grids were examined in a Philips CM-100 transmission electron microscope at 60 kV FEI (Thermo-Fischer, Waltham, MA, The states). 2 contained experiments were carried out and several fields were pictured for each experimental condition. Exosome images were caused with a Gatan Orius 2001 charge-coupled device (CCD, Pleasanton, CA, U.s.) camera.
Digestion of proteins in exosomes and supernatant
The proteins in 80 µl of the supernatant were reduced with 10 mM dithiothreitol (DTT) for xxx min, followed by alkylation with thirty mM iodoacetamide for 60 min at room temperature in the dark. The proteins were digested with i.0 μg LysC-tr (Promega) for 12 h at 37°C and and then diluted and further digested with 1.0 μg trypsin (Promega) for 16 h at 37°C. The digestion was terminated with 0.5% trifluoroacetic acid.
The proteins nowadays in the exosomes were separated from the lipid components by chloroform/methanol precipitation.[31] After resuspension of the chloroform/methanol precipitation pellet in 45 µl of 8 M guanidine, proteins were reduced with 10 mM DTT for 30 min, followed by alkylation with 30 mM iodoacetamide for 60 min in the dark. The sample was diluted ane:1 with fifty mM ammonium bicarbonate. Proteins were digested with one.0 μg LysC-tr for 12 h at 37C and then diluted 4:i with 50 mM NHivHCO3. The proteins were farther digested with 1.0 μg trypsin for xvi h at 37°C, and the digestion stopped with 0.five% trifluoroacetic acid. The peptides were desalted on a reversed-phase SepPak C18 cartridge (Waters, Milford, MA, USA), and eluted with 80% acetonitrile. The eluate was dried in a SpeedVac and the peptides acidified with 2% acetonitrile-0.1% trifluoroacetic acid.
LC-MS/MS assay
A nanoflow UHPLC instrument (Easy nLC, Thermo Fisher Scientific, Waltham, MA, Usa) was coupled on-line to a Q Exactive mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were loaded onto a C18 reversed-phase column (25 cm long, 75 μm inner diameter) and separated with a linear gradient of v–35% buffer B (100% acetonitrile in 0.1% formic acid) at a catamenia rate of 300 nl min–1 over 180 min. MS information were acquired using a information-dependent Top15 method dynamically choosing the most abundant precursor ions from the survey browse (400–1400 1000/z) using HCD fragmentation. Survey scans were caused at a resolution of 70,000 at m/z 400. Unassigned precursor ion accuse states also as singly charged species were excluded from fragmentation. The isolation window was fix to 3 Da and fragmented with normalized collision energies of 27. The maximum ion injection times for the survey scan and the MS/MS scans were 20 ms and lx ms respectively, and the ion target values were gear up to 1E6 and 1e5, respectively. Selected sequenced ions were dynamically excluded for 30 s. Data were caused using Xcalibur software (Thermo-Fishcer, Waltham, MA, U.s.).
Data processing and bioinformatic analysis
Mass spectra were analysed using MaxQuant software version ane.v.2.8 using the Andromeda search engine.[32,33] The initial maximum allowed mass deviation was set to 10 ppm for monoisotopic forerunner ions and 0.v Da for MS/MS peaks. Enzyme specificity was set up to trypsin, defined every bit C-terminal to arginine and lysine excluding proline, and a maximum of two missed cleavages were allowed. Carbamidomethylcysteine was set every bit a fixed modification, Due north-terminal acetylation and methionine oxidation as variable modifications. The spectra were searched with the Andromeda search engine against the Human SWISSPROT sequence database (containing twenty,193 human being protein entries) combined with 248 mutual contaminants and concatenated with the reversed versions of all sequences. Protein identification required at to the lowest degree 1 unique or razor peptide per protein grouping. Quantification in MaxQuant was performed using the built-in XIC-based characterization-free quantification (LFQ) algorithm.[32] The required false positive rate for identification was set to 1% at the peptide level and 1% at the protein level, and the minimum required peptide length was set to six amino acids. Contaminants, reverse identification and proteins only identified past modified peptides were excluded from further data analysis. The LFQ values were log2-transfomed. Afterwards filtering (at least two valid LFQ values in at to the lowest degree one group), the remaining missing LFQ values were imputed from a normal distribution (width 0.iii; down-shift 1.8). A two sample t-examination was used to assess statistical significance of protein abundances using p ≤ 0.05 as the cut-off. For proteomic data analysis including normalization, statistics, hierarchical clustering, and Fisher's exact tests, we used the Perseus platform.[34] The unsupervised hierarchical clustering and heat map were based on protein expression. The rows of the estrus map betoken the proteins, and the columns indicate the samples. The log2 ratios of each protein were z-score normalized for each row. Hierarchical clustering of the z-normalized log2 ratio was performed using Euclidean distances betwixt ways. The number of clusters was set as 300. Genome ontology assay, molecular functions and signalling pathways in differentially expressed proteins was using the GO Slim analysis in the Panther database (http://pantherdb.org/). This nomenclature uses an evolutionary framework to infer protein functions in a species-independent fashion.[35]
Measurement of zeta potential (ζ)
The zeta potentials of BAL exosomes were measured using a ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA) and previously established procedures.[36] It was not necessary to arrange the exosome concentration or the pH of the solution. The first ready of exosomes was measured straight at 18°C. A second sample was stored at +4°C for 4 days. A third sample was frozen at −80°C for four days, allowed to warm upwards to room temperature over 60 min and then measured. Each of these procedures was repeated independently v times.
Western immunoblot
Exosome and cell pellets were lysed in sodium dodecyl sulfate (SDS)-urea lysis buffer [5% SDS, 9 Grand urea, 125 mM Tris HCl pH vi.8 supplemented with protease inhibitor cocktail (Sigma P8340)]. Poly peptide concentration was determined by bicinchoninic acid (BCA, Pierce, Thermo Scientific) and ten μg were dissolved into SDS loading buffer (with 5% βME) and fractionated on 4–15% Mini-protean TGX gels (BioRad, Hercules CA, United states of america) in 1× Tris Glycine SDS (TGS) 1× running buffer at room temperature. Proteins were electro-transferred to PVDF (Immobilon-P, Millipore) in 1× TGS buffer-methanol (20%) buffer. The blots were blocked with 5% milk Tween-20 (0.1%) PBS buffer (T-PBS, pH 7.4) for 1 h and incubated overnight at 4°C in primary antibodies. Antibodies were: anti-CD63 (Abcam ab193349); anti-HSP90 (Prison cell Signaling C45G5, Danvers, MA, USA); anti-Alix/PDC61 (Ab76608); Anti-GRP4 (Jail cell Signaling #2104); and anti- β-actin (Sigma, AC-fifteen). Secondary antibodies were HRP-conjugated anti-rabbit IgG and anti-mouse IgG from Cell Signaling and Southern Biotech (Birmingham, AL, USA), respectively.
Public submission: Nosotros have submitted all relevant data of our experiments to the EV-Rail knowledgebase (EV-Rails ID: {"blazon":"entrez-nucleotide","attrs":{"text":"EV170017","term_id":"151259594","term_text":"EV170017"}}EV170017).[37]
Results
Our analysis of secreted proteins from RNA virus-infected lung epithelial cells indicated innate pathway activation produced secretion of exosomes and changes in the exosome content of over >240 proteins.[x] Seeking to extend these studies to airway in vivo, we isolated exosomes from mice stimulated with synthetic poly (I:C), a price-like receptor (TLR)-3 ligand that mimics the effects of viral replication in a much more reproducible manner.[16,19] In preliminary studies, we observed that freshly prepared BALF exosomes had distinctly dissimilar dynamic low-cal handful (DLS) patterns compared to those frozen at −80°C. Intrigued that storage conditions may underlie this consequence, we conducted a comparative written report using a puddle of enriched exosomes freshly prepared from the BALF. BALF exosomes were prepared using conventional ultracentrifugation, washed in PBS, and analysed right afterward isolation or stored in aliquots at +4°C or at −eighty°C (Figure 1). To confirm exosome enrichment in our training, the fresh exosomal fraction was subjected to Western blot analysis; here we identified the presence of CD63 and Alix (Supplemental Figure one). Stored samples were re-pelleted by ultracentrifugation, and the storage supernatant saved. The fresh and stored exosome samples were compared by DLS backdrop, ultrastructural features, accuse densities and protein contents past LC-MS/MS. To discover proteins lost during leakage, the storage supernatants were compared to the original launder solution from the fresh exosomes by LC-MS/MS (Figure one).
Experimental strategy and assay of exosome size as a function of storage temperature. Schematic of experiment and protein assay. Enriched exosomes isolated via differential centrifugation were prepared, washed, and aliquoted for storage under dissimilar atmospheric condition. Later on storage, samples were re-pelleted, and the pellet (exosome) and storage supernatant assayed. Blue arrows indicate fractions subjected to unbiased LC-MS/MS analyses.
Temperature furnishings on exosome DLS properties
To determine the effect of storage temperature on exosome size, the population distribution for the dissimilar storage conditions was measured by DLS (Figure 2(a)). Freshly prepared exosomes showed an asymmetric size distribution of l–170 nm (Figure 2(a)), with an average size of 94.five ± 1.7 nm (Figure 2(b)). By contrast, exosomes stored at +4°C underwent a shift in average size to 104 ± 1.15 nm (Effigy 2(b)). We also observed a dramatic shift to a Poisson-like distribution in the DLS profile of exosomes stored at −lxxx°C, indicating polydispersity in the size distribution. Multiple overlapping Gaussian size distributions effect in a long tail mixture appearance, suggesting that freezing produced a population of larger nanovesicle aggregates up to 400 nm in diameter (Figure two(a)). This shift was observed in two independent isolations, each measured in triplicate (p < 0.05, t-exam; Supplemental Effigy 2). The average size of the frozen exosomes increased to 125 ± 1.15 nm (Figure 2(b), p < 0.001, t-test). These results were consistent in multiple experiments, and independent of whether the exosomes were thawed slowly on ice or chop-chop at 37°C (not shown).
Effects of storage conditions on exosome size and structure. (a) Dynamic light scattering (DLS) analysis. DLS of enriched exosomes after storage relative to fresh preparations. Shown are intensity plots of size distribution. (b) Average exosome size. Shown is the boilerplate size for the exosome grooming from n = 2 carve up biological replicates. For each indicated temperature, the mean ± SE is shown. * =p < 0.05; *** =p < 0.001. (c) TEM for exosomes as a function of storage temperature. Notation the presence of multi-lamellar membranes in −80°C storage. Images are representative of two independent biological experiments. Additional pictures from these preparations are shown in Supplementary Figure 1.
Temperature effects on exosome ultrastructure
To farther sympathize the changes in DLS, nosotros subjected the exosomes to ultrastructural studies using manual electron microscopy (TEM). Both the freshly prepared exosomes and those stored at +4°C appeared in TEM as isolated, membrane-encapsulated nanovesicles, with the characteristic bogus central depression ("cupping") ascribed to cellulose embedding (Figure ii(c)).[38] By contrast, the exosomes stored at −80°C were larger, aggregated and showed the appearance of multi-lamellar membrane layers, consistent with the DLS study (additional images are shown in Supplemental Figure 3).
Storage effects on exosome zeta potential (ζ)
We next examined the result of storage weather on the charge density distribution around the exosome, a parameter known as the zeta potential (ζ) that is virtually independent of temperature. Freshly prepared BALF exosomes maintained at 18°C demonstrated ζ between −34.eight and −32.4 mV. Individual tracings are shown in Figure three(a), and boilerplate ζ values shown in Figure 3(b). These ζ values are within the potential range expected for airway exosomes due to the high distribution of negatively charged membrane phospholipids.[39] It is remarkable that afterwards thawing from −80°C, the ζ was farther diminished to −sixteen.5 mV to −9.88 mV, indicating that the freezing process is extremely confusing to the exosome construction and physical backdrop (Effigy 3(a), 3(b)). Importantly, at such ζ, the exosomes possess near no barrier against fusion processes, providing a physicochemical caption for the exosome fusion observed in TEM and increased size characterized by DLS. Interestingly, these findings are in understanding with the known structural changes induced by freezing liposomes.[40,41]
Zeta potential measurements. (a) Individual tracings of BALF exosomes that were stored at + 18°C (fresh), + iv°C or –80°C followed by thawing every bit indicated. (b) Private zeta potentials of BAL-derived exosomes for 5 individual measurements. Notation the progressive loss of ζ with storage.
Effects of storage weather condition on exosome poly peptide content
To establish a baseline of the exosome content, we conducted unbiased proteomic profiling using LC-MS/MS after lipid depletion using an optimized chloroform/methanol precipitation method.[31] Nosotros identified a full of 1140 proteins in freshly isolated exosomes at an FDR of 1% or less. GO Slim assay of signalling pathways showed significant enrichment of "plasminogen activating pour", "cell wheel" and "ubiquitin protein deposition" pathways (Effigy four(a)). Glycolysis and protein translation were identified as the most enriched biological processes in the exosome fractions (Figure 4(b)). The name, identifier, GO annotations, and the fold modify of the abundance of the proteins identified are listed in Supplemental Table S1.
Unbiased proteomics of exosome content in fresh preparations. (a) Become Slim assay of signalling pathways. A full of 1140 proteins identified in freshly isolated exosomes were mapped to thirteen pathways. Confined indicate signalling pathway enrichment (%) over reference human proteome (lower Ten axis). For each signalling pathway, the p-value of the enrichment is indicated by black dot (top Ten centrality). (b) Go Slim analysis of biological processes. Data are presented as in Figure 4(a). Reddish confined, biological processes enriched in exosomal fraction; blueish, processes depleted.
Fresh vs. +4°C exosomes
To decide whether the different storage atmospheric condition affected the exosome protein content, we conducted pairwise comparisons of the freshly isolated exosomes vs. those at +4°C and −80°C storage using characterization-free LC-MS/MS (Figure 1). Freshly prepared duplicate biological replicates were kickoff profiled vs. exosomes stored at +four°C (representing three contained preparations in two separate biological replicates). In this analysis, differential protein expression was identified using a volcano plot, where −log10 transformed p-value (of 2 sample t-test) is plotted vs. the fold modify in protein abundance of exosomes of the 2 dissimilar storage conditions (fresh vs. +4°C, Figure 5(a)). From this analysis, 544 (47%) of proteins showed no divergence in abundance in the exosomes as a result of the storage. Nonetheless, 457 proteins were changed after +4°C relative to fresh exosomes; of these, 312 were depleted in the exosomes stored at +4°C (Effigy 5(a); log2 normalized expression profiles are clustered in Figure 5(c)) (Supplemental Table S1).
Effect of storage temperatures on exosome protein content. Volcano plots of (a) fresh vs. +4°C; (b) fresh vs. −fourscore°C. (c) Heatmap of significantly changed proteins (fresh vs. +4°C). Log 2 poly peptide abundance was Z-score normalized and subjected to hierarchical clustering. Each row is a protein; columns represent separate experimental replicates. Red, higher level of expression; green, depression level of expression. (d) Heatmap of significantly changed proteins (fresh vs. −80°C).
Fresh vs. −80°C exosomes
A similar comparison was performed for freshly isolated exosomes vs. those frozen at −80°C. From this analysis, 527 (46%) of proteins showed no departure in abundance every bit a issue of the storage, and 315 proteins were depleted in exosomes after −eighty°C storage (Figure 5(b), five(d) (Supplemental Tabular array S1).
The two groups of storage-depleted proteins were highly overlapping, with 262 proteins in common. Interestingly, these proteins were not statistically enriched in a unmarried Go molecular function, indicating that these storage-labile proteins affect a wide variety of biological processes and molecular functions.
+iv°C vs. −eighty°C exosomes
Finally, differential comparison was besides performed for exosomes stored at +4°C vs. −eighty°C (Figure 6(a)). A total of 848 high-confidence exosome proteins were identified (Table 1). This protein set contained fourscore of the top 100 exosome proteins referred in Exocarta and the fractions were enriched in cytoplasmic, ribosomal and vesicular proteins (Table ii). From this analysis, 756 (89%) of proteins showed no difference in affluence every bit a issue of the storage temperature. However, 61 proteins were depleted afterward +four°C storage (or more abundant in the exosomes stored at −80°C); past contrast, 31 proteins were more abundant in the exosomes stored at +4°C relative to those stored at −eighty°C, indicating that a small population of exosome proteins was more than sensitive to the storage temperature (Effigy vi(b)). Consistency in the changes in protein abundance by replicate was analysed past hierarchical clustering of the log2-normalized abundance (Figure 6(b)). A comparison of proteins identified from the +4°C and −eighty°C storage temperatures is shown by a pie diagram (Figure vi(c)). The name, identifier, Go annotations, and the fold alter of the abundance of the proteins identified are listed in Supplemental Table S2.
Tabular array i.
Exosomal proteins identified in BALF exosomes. A full of 848 high-confidence exosome proteins identified in stored samples were compared to the Exocarta database. Of the acme 100 exosomal proteins, 80 were matched. For each matched protein is listed the gene name, Uniprot identification number (#), characteristics of identification and quantification of changes in abundance past storage condition. Cherry indicates enrichment and dark-green depletion by comparing of the two storage conditions.
| Protein names | Cistron names | Uniprot | Identification | Quantification | ||||
|---|---|---|---|---|---|---|---|---|
| Razor + unique peptides | Q value | Score | t-test significant | p-value -log10 | Log2 fold change 4°C vs. −80°C | |||
| Serum albumin | Alb | {"blazon":"entrez-poly peptide","attrs":{"text":"P07724","term_id":"5915682","term_text":"P07724"}}P07724 | 91 | 0 | 323.31 | + | 1.54 | 0.82 |
| Myosin-9 | Myh9 | {"type":"entrez-poly peptide","attrs":{"text":"Q8VDD5","term_id":"205371802","term_text":"Q8VDD5"}}Q8VDD5 | 125 | 0 | 323.31 | + | 2.60 | 0.23 |
| Heat shock protein HSP 90-beta | Hsp90ab1 | {"type":"entrez-protein","attrs":{"text":"P11499","term_id":"341941065","term_text":"P11499"}}P11499 | 33 | 0 | 323.31 | + | ane.51 | −0.34 |
| Programmed jail cell expiry 6-interacting poly peptide | Pdcd6ip | {"blazon":"entrez-poly peptide","attrs":{"text":"Q9WU78","term_id":"341942206","term_text":"Q9WU78"}}Q9WU78 | xxx | 0 | 323.31 | + | ane.83 | −0.36 |
| Filamin-A | Flna | {"type":"entrez-protein","attrs":{"text":"Q8BTM8","term_id":"341941149","term_text":"Q8BTM8"}}Q8BTM8 | 89 | 0 | 323.31 | + | two.11 | −0.41 |
| Tubulin alpha-1B chain | Tuba1b | {"type":"entrez-protein","attrs":{"text":"P05213","term_id":"55977764","term_text":"P05213"}}P05213 | 34 | 0 | 323.31 | + | one.89 | −0.48 |
| T-circuitous protein i subunit alpha | Tcp1 | {"type":"entrez-protein","attrs":{"text":"P11983","term_id":"1729865","term_text":"P11983"}}P11983 | 29 | 0 | 297.26 | + | 3.61 | −0.49 |
| Heat shock poly peptide HSP 90-blastoff | Hsp90aa1 | {"blazon":"entrez-protein","attrs":{"text":"P07901","term_id":"1170384","term_text":"P07901"}}P07901 | 13 | 0 | 152.25 | + | 1.34 | −0.lxx |
| Importin subunit beta-1 | Kpnb1 | {"type":"entrez-protein","attrs":{"text":"P70168","term_id":"341940828","term_text":"P70168"}}P70168 | 9 | 0 | 41.415 | + | ane.62 | −0.71 |
| Ubiquitin-similar modifier-activating enzyme 1 | Uba1 | {"type":"entrez-protein","attrs":{"text":"Q02053","term_id":"267190","term_text":"Q02053"}}Q02053 | 22 | 0 | 196.07 | + | 2.02 | −0.89 |
| Rab GDP dissociation inhibitor beta | Gdi2 | Q61598-2 | 9 | 0 | 42.816 | + | 1.63 | −1.06 |
| Ras-related C3 botulinum toxin substrate i | Rac1 | {"type":"entrez-protein","attrs":{"text":"P63001","term_id":"51702788","term_text":"P63001"}}P63001 | iv | 0 | 22.516 | 1.24 | 1.68 | |
| Actin, cytoplasmic i | Actb | {"type":"entrez-protein","attrs":{"text":"P60710","term_id":"46397334","term_text":"P60710"}}P60710 | i | 0 | 75.629 | 0.87 | 1.32 | |
| Fructose-bisphosphate aldolase A | Aldoa | {"blazon":"entrez-protein","attrs":{"text":"P05064","term_id":"113607","term_text":"P05064"}}P05064 | 11 | 0 | 219.1 | 0.53 | 0.96 | |
| Triosephosphate isomerase | Tpi1 | {"type":"entrez-protein","attrs":{"text":"P17751","term_id":"1915398945","term_text":"P17751"}}P17751 | 10 | 0 | 75.079 | 0.36 | 0.66 | |
| Peroxiredoxin-i | Prdx1 | {"type":"entrez-protein","attrs":{"text":"P35700","term_id":"547923","term_text":"P35700"}}P35700 | 13 | 0 | 119.26 | 0.19 | 0.35 | |
| Annexin A5 | Anxa5 | {"blazon":"entrez-protein","attrs":{"text":"P48036","term_id":"1351942","term_text":"P48036"}}P48036 | 24 | 0 | 323.31 | 0.46 | 0.33 | |
| Adenosylhomocysteinase | Ahcy | {"type":"entrez-protein","attrs":{"text":"P50247","term_id":"21431841","term_text":"P50247"}}P50247 | 2 | 0 | 4.9712 | 0.32 | 0.31 | |
| EH domain-containing poly peptide iv | Ehd4 | {"blazon":"entrez-poly peptide","attrs":{"text":"Q9EQP2","term_id":"18202868","term_text":"Q9EQP2"}}Q9EQP2 | eight | 0 | 22.779 | 0.40 | 0.30 | |
| Ras-related protein Rab-1A | Rab1A | {"type":"entrez-protein","attrs":{"text":"P62821","term_id":"51338604","term_text":"P62821"}}P62821 | ii | 0 | xiv.628 | 0.17 | 0.23 | |
| xiv-3-3 protein theta | Ywhaq | P68254-ii | 5 | 0 | 18.931 | 0.12 | 0.twenty | |
| Blastoff-2-macroglobulin | A2m | {"type":"entrez-protein","attrs":{"text":"Q61838","term_id":"338817897","term_text":"Q61838"}}Q61838 | 94 | 0 | 323.31 | 0.18 | 0.18 | |
| Actin, cytoplasmic two | Actg1 | {"type":"entrez-protein","attrs":{"text":"P63260","term_id":"54036677","term_text":"P63260"}}P63260 | 49 | 0 | 323.31 | 0.38 | 0.18 | |
| Clathrin heavy chain ane | Cltc | {"type":"entrez-poly peptide","attrs":{"text":"Q68FD5","term_id":"66773801","term_text":"Q68FD5"}}Q68FD5 | 42 | 0 | 323.31 | 0.36 | 0.17 | |
| Histone H4 | Hist1h4a | {"type":"entrez-protein","attrs":{"text":"P62806","term_id":"51317340","term_text":"P62806"}}P62806 | 24 | 0 | 323.31 | 0.18 | 0.14 | |
| Annexin A2 | Anxa2 | {"type":"entrez-poly peptide","attrs":{"text":"P07356","term_id":"113951","term_text":"P07356"}}P07356 | 20 | 0 | 323.31 | 0.24 | 0.12 | |
| Guanine nucleotide-bounden protein Grand(I)/G(S)/One thousand(T) subunit beta-ane | Gnb1 | {"type":"entrez-protein","attrs":{"text":"P62874","term_id":"51317303","term_text":"P62874"}}P62874 | 6 | 0 | 57.544 | 0.28 | 0.10 | |
| 50-lactate dehydrogenase A concatenation | Ldha | {"blazon":"entrez-poly peptide","attrs":{"text":"P06151","term_id":"126048","term_text":"P06151"}}P06151 | 12 | 0 | 93.185 | 0.07 | 0.05 | |
| 78 kDa glucose-regulated poly peptide | Hspa5 | {"blazon":"entrez-protein","attrs":{"text":"P20029","term_id":"2506545","term_text":"P20029"}}P20029 | 14 | 0 | eighty.842 | 0.01 | 0.03 | |
| Guanine nucleotide-binding poly peptide G(i) subunit alpha-ii | Gnai2 | {"blazon":"entrez-protein","attrs":{"text":"P08752","term_id":"341941148","term_text":"P08752"}}P08752 | 8 | 0 | 55.519 | 0.07 | 0.03 | |
| Sodium/potassium-transporting ATPase subunit alpha-1 | Atp1a1 | {"type":"entrez-poly peptide","attrs":{"text":"Q8VDN2","term_id":"55976751","term_text":"Q8VDN2"}}Q8VDN2 | 14 | 0 | 52.724 | 0.05 | 0.03 | |
| Cofilin-i | Cfl1 | {"type":"entrez-protein","attrs":{"text":"P18760","term_id":"116849","term_text":"P18760"}}P18760 | 20 | 0 | 323.31 | 0.05 | 0.02 | |
| Ras-related protein Rap-1b | Rap1b | {"blazon":"entrez-protein","attrs":{"text":"Q99JI6","term_id":"47117288","term_text":"Q99JI6"}}Q99JI6 | 11 | 0 | 121.29 | 0.07 | 0.01 | |
| Profilin-ane | Pfn1 | {"type":"entrez-protein","attrs":{"text":"P62962","term_id":"51702779","term_text":"P62962"}}P62962 | 17 | 0 | 323.31 | 0.02 | −0.01 | |
| T-complex poly peptide one subunit epsilon | Cct5 | {"type":"entrez-protein","attrs":{"text":"P80316","term_id":"549058","term_text":"P80316"}}P80316 | 25 | 0 | 230.38 | 0.02 | −0.03 | |
| Lactadherin | Mfge8 | P21956-2 | 19 | 0 | 254.73 | 0.07 | −0.05 | |
| T-circuitous poly peptide 1 subunit gamma | Cct3 | {"type":"entrez-protein","attrs":{"text":"P80318","term_id":"549059","term_text":"P80318"}}P80318 | 30 | 0 | 323.31 | 0.14 | −0.06 | |
| Lysosome-associated membrane glycoprotein 2 | Lamp2 | P17047-3 | 3 | 0 | half dozen.5452 | 0.04 | −0.07 | |
| Elongation factor 1-alpha 1 | Eef1a1 | {"blazon":"entrez-poly peptide","attrs":{"text":"P10126","term_id":"56405010","term_text":"P10126"}}P10126 | 20 | 0 | 323.31 | 0.17 | −0.10 | |
| Transketolase | Tkt | {"type":"entrez-poly peptide","attrs":{"text":"P40142","term_id":"730956","term_text":"P40142"}}P40142 | 35 | 0 | 323.31 | 0.31 | −0.13 | |
| Glyceraldehyde-3-phosphate dehydrogenase | Gapdh | {"type":"entrez-protein","attrs":{"text":"P16858","term_id":"120702","term_text":"P16858"}}P16858 | 19 | 0 | 323.31 | 0.32 | −0.13 | |
| T-complex poly peptide 1 subunit beta | Cct2 | {"type":"entrez-protein","attrs":{"text":"P80314","term_id":"22654291","term_text":"P80314"}}P80314 | 41 | 0 | 323.31 | 0.41 | −0.xiv | |
| Syntenin-1 | Sdcbp | {"type":"entrez-poly peptide","attrs":{"text":"O08992","term_id":"20455282","term_text":"O08992"}}O08992 | 11 | 0 | 236.88 | 0.39 | −0.xiv | |
| Moesin | Msn | {"blazon":"entrez-protein","attrs":{"text":"P26041","term_id":"462608","term_text":"P26041"}}P26041 | 44 | 0 | 323.31 | 0.12 | −0.16 | |
| Elongation gene 2 | Eef2 | {"type":"entrez-protein","attrs":{"text":"P58252","term_id":"18202285","term_text":"P58252"}}P58252 | 30 | 0 | 247.09 | one.09 | −0.18 | |
| Galectin-3-bounden protein | Lgals3bp | {"type":"entrez-poly peptide","attrs":{"text":"Q07797","term_id":"81882251","term_text":"Q07797"}}Q07797 | 27 | 0 | 323.31 | 0.25 | −0.22 | |
| GTP-binding nuclear protein Ran | Ran | {"type":"entrez-protein","attrs":{"text":"P62827","term_id":"51338599","term_text":"P62827"}}P62827 | 11 | 0 | 169.36 | 0.61 | −0.23 | |
| ADP-ribosylation cistron 1 | Arf1 | {"type":"entrez-protein","attrs":{"text":"P84078","term_id":"51316986","term_text":"P84078"}}P84078 | eight | 0 | 51.639 | 0.43 | −0.24 | |
| fourteen–3-3 protein epsilon | Ywhae | {"type":"entrez-poly peptide","attrs":{"text":"P62259","term_id":"60391192","term_text":"P62259"}}P62259 | seven | 0 | 73.25 | 0.14 | −0.24 | |
| Phosphoglycerate kinase 1 | Pgk1 | {"type":"entrez-protein","attrs":{"text":"P09411","term_id":"146345481","term_text":"P09411"}}P09411 | 22 | 0 | 323.31 | 0.86 | −0.26 | |
| Major vault poly peptide | Mvp | {"type":"entrez-poly peptide","attrs":{"text":"Q9EQK5","term_id":"341941144","term_text":"Q9EQK5"}}Q9EQK5 | 56 | 0 | 323.31 | 0.nineteen | −0.27 | |
| Pyruvate kinase PKM | Pkm | {"type":"entrez-protein","attrs":{"text":"P52480","term_id":"146345448","term_text":"P52480"}}P52480 | 54 | 0 | 323.31 | 0.75 | −0.28 | |
| Transitional endoplasmic reticulum ATPase | Vcp | {"type":"entrez-poly peptide","attrs":{"text":"Q01853","term_id":"146291078","term_text":"Q01853"}}Q01853 | 54 | 0 | 323.31 | ane.11 | −0.32 | |
| Ras-related protein Rab-7a | Rab7a | {"blazon":"entrez-protein","attrs":{"text":"P51150","term_id":"46397834","term_text":"P51150"}}P51150 | xiii | 0 | 323.31 | 0.34 | −0.33 | |
| Annexin A11 | Anxa11 | {"type":"entrez-protein","attrs":{"text":"P97384","term_id":"341940228","term_text":"P97384"}}P97384 | 13 | 0 | 86.482 | 0.39 | −0.34 | |
| Guanine nucleotide-binding poly peptide 1000(I)/G(S)/G(T) subunit beta-ii | Gnb2 | {"type":"entrez-protein","attrs":{"text":"P62880","term_id":"51317305","term_text":"P62880"}}P62880 | 12 | 0 | 121.xvi | 0.73 | −0.34 | |
| Heat shock lxx kDa protein 1A | Hspa1a | {"type":"entrez-protein","attrs":{"text":"Q61696","term_id":"56757667","term_text":"Q61696"}}Q61696 | 9 | 0 | 37.922 | 0.23 | −0.34 | |
| Fat acid synthase | Fasn | {"type":"entrez-poly peptide","attrs":{"text":"P19096","term_id":"54040727","term_text":"P19096"}}P19096 | 29 | 0 | 141.38 | 1.27 | −0.38 | |
| Cell segmentation control protein 42 homologue | Cdc42 | {"type":"entrez-protein","attrs":{"text":"P60766","term_id":"322510016","term_text":"P60766"}}P60766 | viii | 0 | 125.08 | 0.66 | −0.38 | |
| Transforming protein RhoA | Rhoa | {"type":"entrez-protein","attrs":{"text":"Q9QUI0","term_id":"13633865","term_text":"Q9QUI0"}}Q9QUI0 | 9 | 0 | 112.21 | 0.26 | −0.39 | |
| Peroxiredoxin-2 | Prdx2 | {"type":"entrez-protein","attrs":{"text":"Q61171","term_id":"2499469","term_text":"Q61171"}}Q61171 | iv | 0 | six.8609 | 0.46 | −0.41 | |
| Annexin A4 | Anxa4 | {"type":"entrez-protein","attrs":{"text":"P97429","term_id":"341940625","term_text":"P97429"}}P97429 | 15 | 0 | 134.02 | 0.36 | −0.44 | |
| Heat shock cognate 71 kDa protein | Hspa8 | {"type":"entrez-protein","attrs":{"text":"P63017","term_id":"51702275","term_text":"P63017"}}P63017 | 46 | 0 | 323.31 | 1.23 | −0.45 | |
| Alpha-enolase | Eno1 | {"type":"entrez-protein","attrs":{"text":"P17182","term_id":"13637776","term_text":"P17182"}}P17182 | 35 | 0 | 323.31 | 0.32 | −0.49 | |
| Chloride intracellular channel protein 1 | Clic1 | {"type":"entrez-protein","attrs":{"text":"Q9Z1Q5","term_id":"6685328","term_text":"Q9Z1Q5"}}Q9Z1Q5 | 17 | 0 | 187.54 | 1.08 | −0.49 | |
| 14-3-3 protein beta/alpha | Ywhab | Q9CQV8-two | v | 0 | 25.722 | 0.twoscore | −0.51 | |
| Ras-related protein Rab-5C | Rab5c | {"type":"entrez-protein","attrs":{"text":"P35278","term_id":"38258917","term_text":"P35278"}}P35278 | vi | 0 | 61.235 | 0.24 | −0.54 | |
| 14-3-three protein zeta/delta | Ywhaz | {"type":"entrez-protein","attrs":{"text":"P63101","term_id":"52000885","term_text":"P63101"}}P63101 | 13 | 0 | 186.xiv | 0.70 | −0.55 | |
| Annexin A1 | Anxa1 | {"type":"entrez-protein","attrs":{"text":"P10107","term_id":"113945","term_text":"P10107"}}P10107 | 27 | 0 | 323.31 | 0.85 | −0.58 | |
| Ras-related protein Rab-14 | Rab14 | {"type":"entrez-protein","attrs":{"text":"Q91V41","term_id":"46577103","term_text":"Q91V41"}}Q91V41 | 9 | 0 | 72.052 | 0.59 | −0.59 | |
| ATP-citrate synthase | Acly | {"blazon":"entrez-protein","attrs":{"text":"Q91V92","term_id":"21263374","term_text":"Q91V92"}}Q91V92 | 23 | 0 | 197.13 | 0.78 | −0.63 | |
| Thrombospondin-1 | Thbs1 | {"type":"entrez-protein","attrs":{"text":"P35441","term_id":"549134","term_text":"P35441"}}P35441 | 7 | 0 | 34.905 | 0.40 | −0.65 | |
| Ezrin | Ezr | {"blazon":"entrez-poly peptide","attrs":{"text":"P26040","term_id":"32363497","term_text":"P26040"}}P26040 | 24 | 0 | 323.31 | 0.20 | −0.66 | |
| Erythrocyte band 7 integral membrane poly peptide | Stom | {"type":"entrez-protein","attrs":{"text":"P54116","term_id":"122066246","term_text":"P54116"}}P54116 | 3 | 0 | 14.57 | 0.99 | −0.74 | |
| L-lactate dehydrogenase B chain | Ldhb | {"type":"entrez-poly peptide","attrs":{"text":"P16125","term_id":"126042","term_text":"P16125"}}P16125 | 3 | 0 | seven.6375 | 0.22 | −0.76 | |
| Alpha-actinin-4 | Actn4 | {"type":"entrez-protein","attrs":{"text":"P57780","term_id":"13123946","term_text":"P57780"}}P57780 | seven | 0 | 46.719 | 0.66 | −0.81 | |
| Pyruvate kinase PKM | Pkm | P52480-2 | 2 | 0 | 11.758 | 0.79 | −0.84 | |
| Annexin A6 | Anxa6 | {"blazon":"entrez-poly peptide","attrs":{"text":"P14824","term_id":"341940596","term_text":"P14824"}}P14824 | 17 | 0 | 94.565 | 0.72 | −0.88 | |
| Tubulin alpha-1A chain | Tuba1a | {"type":"entrez-poly peptide","attrs":{"text":"P68369","term_id":"55977479","term_text":"P68369"}}P68369 | iii | 0 | 66.81 | 0.88 | −0.95 | |
| Peptidyl-prolyl cis-trans isomerase A | Ppia | {"type":"entrez-protein","attrs":{"text":"P17742","term_id":"118105","term_text":"P17742"}}P17742 | 14 | 0 | 161.63 | 0.79 | −ane.11 | |
Table two.
Subcellular compartment enrichment of exosomal proteins. A total of 848 high-confidence exosome proteins from stored samples were analysed for subcellular component by GO-Slim (Panther database). Shown are the fold enrichment of the pathway and the significance (p-value, Bonferroni correction).
| PANTHER GO-Slim cellular component | Enrichment | p-value |
|---|---|---|
| Ribosome (GO:0005840) | 12.64 | 1.09E-36 |
| Cytosol (GO:0005829) | vii.four | 9.33E-40 |
| Vesicle coat (GO:0030120) | six.53 | 2.49E-03 |
| Ribonucleoprotein complex (Become:0030529) | 6.16 | seven.39E-29 |
| Actin cytoskeleton (GO:0015629) | 4.43 | viii.50E-x |
| Macromolecular complex (Become:0032991) | two.7 | 2.40E-21 |
| Cytoskeleton (Become:0005856) | ii.47 | 2.05E-06 |
| Cytoplasm (GO:0005737) | 2.43 | 1.58E-23 |
| Extracellular space (Become:0005615) | 2.26 | 4.94E-05 |
| Organelle (GO:0043226) | ii.07 | eight.58E-19 |
| Intracellular (GO:0005622) | 1.92 | 1.34E-23 |
| Jail cell office (GO:0044464) | one.89 | 2.08E-22 |
| Extracellular region (Get:0005576) | i.83 | ane.87E-03 |
| Membrane (Become:0016020) | 0.36 | 9.08E-11 |
| Plasma membrane (GO:0005886) | 0.32 | 2.36E-07 |
| Integral to membrane (GO:0016021) | < 0.2 | 4.91E-fifteen |
Furnishings of storage condition on exosome protein content. Unbiased proteomics using label-free LC-MS/MS was conducted on BALF exosomes in six individual samples from ii biological replicates. (a) Volcano plot for differentially abundant proteins. Shown is a volcano plot of the differential protein expression. Y axis, – log10 transformed p-value of two sample; X axis, the fold change in protein affluence (+iv°C relative to −80°C). (b) Hierarchical clustering as in Effigy 4. (c) Pie chart of differentially expressed proteins. (d) Left, GO Slim analysis of biological pathways for 61 proteins depleted from exosomes by storage at 4°C. Each pathway is displayed by % enrichment (bar) and p-value (scatter plot). Correct, signalling pathway enrichment. (e) Left, GO analysis of 31 proteins depleted from exosomes by storage at −80°C. Right, signalling pathway enrichment.
The 61 proteins more than abundant in the −80°C storage relative to the +four°C storage status were farther analysed for by Go Slim for biological process and signalling pathways. The top 3 biological processes of 17 that were enriched included "neurotransmitter secretion", "JNK pour" and "chromosomal segregation" (Figure 6(d), left). "O-antigen biosynthesis" was the top-ranked signalling pathway (Figure half-dozen(d), right).
Conversely, 31 proteins were more arable at +4°C relative to −fourscore°C storage conditions, indicating their depletion by storage at −lxxx°C (Effigy 6(b)). The superlative two biological processes of the eight enriched processes were "antigen presentation" and "gluconeogenesis" (Effigy 6(e), left). Ii of the meridian signalling pathways identified were "pentose pathway" and "vitamin D metabolism" (Effigy six(east), right). These data indicate that exosome storage volition not only affect vesicular structure and charge density, simply also the biological role of its contents.
Protein dissociation and leakage from exosomes into the supernatant upon storage
To farther understand the effects of storage, we used LC-MS/MS to identify and quantify the proteins in the supernatant afterward +iv°C and −80°C storage (Effigy 1). A full of 699 proteins were identified with high confidence, and functionally analysed past Become nomenclature (Supplemental Tabular array S3). Interestingly, these proteins affect biological pathways that are functionally distinct from those identified in the exosome grooming. For example, the supernatant proteins are enriched in carbohydrate metabolism (gluconeogenesis/glycolysis), TCA cycle and fatty acid biosynthesis pathways (Table iii). These proteins were compared for differential expression past plotting the – log10 transformed p-value vs. the fold change of protein affluence in the volcano plot shown in Figure seven(a). We noted that the expression of 554 proteins is unchanged, appearing in the supernatant independently on the storage conditions. Consistency in the changes in protein affluence by replicate was analysed by hierarchical clustering of the log2-normalized abundance (Figure7(b)), Interestingly, a smaller group of proteins appeared in the supernatants depending on the storage weather; 67 proteins were enriched in the supernatant from the exosomes stored at −80°C, and a set up of 78 unique proteins was enriched in the supernatant from the exosomes stored at +four°C (p < 0.05) (Figure 7(c)).
Table 3.
Go analysis of leak/peri-exosomal proteins. A full of 699 high-confidence proteins actualization in the +four°C and −80°C storage supernatant were analysed for biological processes past Get-Slim (Panther database). Shown are the fold enrichment of the biological process and the significance (p-value, Bonferroni correction).
| PANTHER GO-Slim biological process | Enrichment | p-value |
|---|---|---|
| Gluconeogenesis (GO:0006094) | 10.99 | five.44E-03 |
| Tricarboxylic acid cycle (Become:0006099) | 10.45 | i.57E-03 |
| Glycolysis (GO:0006096) | 9.8 | 1.28E-04 |
| Fatty acid biosynthetic process (Get:0006633) | 6.3 | 1.53E-03 |
| Purine nucleobase metabolic process (Get:0006144) | 6.28 | ane.90E-04 |
| Protein complex associates (Get:0006461) | five.sixteen | 4.86E-07 |
| Protein complex biogenesis (GO:0070271) | five.13 | 5.40E-07 |
| Blood coagulation (Go:0007596) | 4.62 | 9.15E-03 |
| Monosaccharide metabolic process (Get:0005996) | iv.ii | ane.01E-02 |
| Chromatin organization (GO:0006325) | 4.04 | 2.32E-07 |
| Macrophage activation (GO:0042116) | three.96 | iii.51E-02 |
| Fatty acid metabolic process (Become:0006631) | iii.83 | 7.00E-05 |
| Translation (GO:0006412) | 3.64 | 1.18E-06 |
| Proteolysis (GO:0006508) | iii.59 | 1.14E-13 |
| Cellular component biogenesis (Go:0044085) | iii.45 | 2.17E-09 |
| Cellular amino acid metabolic process (Get:0006520) | 3.34 | 5.79E-04 |
| Generation of precursor metabolites and energy (Become:0006091) | iii.22 | 2.96E-03 |
| Catabolic procedure (GO:0009056) | 2.98 | 7.38E-xi |
| Lipid metabolic process (GO:0006629) | 2.78 | 1.97E-06 |
| Cellular component organisation or biogenesis (GO:0071840) | 2.58 | 4.22E-eighteen |
| Cellular component arrangement (GO:0016043) | 2.45 | four.05E-14 |
| Cellular component morphogenesis (GO:0032989) | 2.21 | 2.02E-02 |
| Organelle organization (GO:0006996) | two.15 | 1.30E-03 |
| Protein metabolic process (GO:0019538) | 2.12 | three.65E-12 |
| Allowed system process (GO:0002376) | 1.79 | iii.12E-03 |
| Primary metabolic process (GO:0044238) | 1.69 | 2.39E-18 |
| Transport (Go:0006810) | 1.61 | iii.94E-03 |
| Metabolic procedure (Get:0008152) | 1.58 | 2.75E-16 |
| Localization (Go:0051179) | ane.55 | nine.11E-03 |
| Cellular process (Become:0009987) | 1.24 | 1.05E-03 |
| Multicellular organismal process (GO:0032501) | 0.58 | 2.03E-02 |
| Unclassified (UNCLASSIFIED) | 0.57 | 0.00E+00 |
| RNA metabolic procedure (GO:0016070) | 0.53 | 1.81E-02 |
| Developmental procedure (GO:0032502) | 0.v | 7.71E-03 |
| Cell surface receptor signalling pathway (Get:0007166) | 0.42 | 4.02E-03 |
| Neurological arrangement process (Become:0050877) | 0.34 | four.45E-05 |
| Transcription from RNA polymerase II promoter (Become:0006366) | 0.27 | ii.92E-04 |
| Regulation of transcription from RNA polymerase Two promoter (GO:0006357) | 0.25 | 3.13E-03 |
| Sensory perception (GO:0007600) | 0.25 | 7.18E-05 |
| 1000-protein coupled receptor signalling pathway (GO:0007186) | 0.25 | i.22E-02 |
| Transcription, Dna-dependent (Become:0006351) | 0.21 | 3.82E-07 |
Effect of storage condition on leakage and dissociation of peri-exosomal proteins. Unbiased proteomics using label-free LC-MS/MS were conducted on peri-exosomal proteins (supernatants later on storage) of six individual samples from two biological replicates. (a) Volcano plot for proteins differentially expressed. Y axis, – log10 transformed p-value of two samples; the 10 centrality is the fold change in protein abundance. (b) Hierarchical clustering. Log 2 protein abundance was Z-score normalized and subjected to hierarchical clustering. Each row is a protein; columns correspond split up experimental replicates. (c) Pie nautical chart of differentially expressed proteins.
We adjacent analysed the supernatant fractions for the presence of the 61 proteins depleted from the exosome preparations stored at +4°C. Nosotros found that 22 proteins depleted during +4°C storage did not announced in the supernatant. We interpret these data to mean that these proteins were metabolized or degraded at +4°C. Conversely, 29 of the 31 proteins depleted from the exosome preparations stored at −80°C appeared in the soluble supernatant. These proteins included cytokines (CXCL15, CC10) and serine proteases (Serpina-1c and -1d, shown in estrus map in Figure 6(b)). Nosotros translate this finding to indicate that the majority of the proteins lost at −fourscore°C leaked into the supernatant due to membrane fusion and/or membrane disruption.
Identification of proteins in the supernatant during storage
To further exclude the possibility that the proteins appearing in the storage supernatants were due to nonspecific contamination, we conducted a quantitative proteomic analysis of the "launder" sample from the 100,000 × g centrifugation (Figure i). By "volcano plot" analysis, the protein abundance of 224 proteins were significantly increased in the exosome supernatant afterward +4°C storage relative to the starting wash buffer (Figure 8(a), Tabular array iv, Supplemental Tabular array S4), conspicuously demonstrating that these proteins were not due to carryover from inadequate washing. These proteins correspond a combination of: (1) proteins initially tightly associated with exosomes later dissociate during storage; (two) proteins that are secreted during storage; or (3) proteins that are released due to exosome lysis/leakage. It is important to notation that the biological processes of these supernatant proteins are distinct from those of the parent exosome, arguing against nonspecific exosome leakage. For instance the top 2 enriched biological processes were "transcriptional regulation" and "chromatin system" (compare Table four and Supplemental Tabular array S4 with Table 3). Every bit discussed farther on, we suggest that the near cogent explanation is that these proteins represent dissociated, "peri-exosomal" proteins.
Table iv.
Biological processes of leak/peri-exosomal proteins enriched at iv°C storage. A total of 224 loftier-confidence proteins enriched in the supernatant afterwards 4°C storage were analysed for biological processes by Get-Slim (Panther database). Shown are the fold enrichment of the pathway and the p-value (Bonferroni correction).
| PANTHER GO-Slim biological procedure | Enrichment | p-value |
|---|---|---|
| Transcription initiation from RNA polymerase Two promoter (Get:0006367) | xi.63 | 1.98E-02 |
| Chromatin organization (GO:0006325) | 10 | 1.52E-14 |
| Protein complex assembly (GO:0006461) | 8.51 | 6.49E-06 |
| Protein complex biogenesis (Become:0070271) | 8.46 | 6.93E-06 |
| Translation (Get:0006412) | five.79 | half dozen.35E-06 |
| Cellular component biogenesis (Go:0044085) | 5.21 | 9.74E-08 |
| Organelle organization (Become:0006996) | iv.55 | 1.35E-09 |
| Cellular component organization or biogenesis (Become:0071840) | 4.xv | 1.01E-20 |
| Cellular component organization (GO:0016043) | 3.86 | 7.44E-16 |
| Proteolysis (GO:0006508) | 3.33 | two.44E-03 |
| Catabolic process (GO:0009056) | 2.97 | 3.98E-03 |
| Protein metabolic process (GO:0019538) | 2.52 | 5.65E-07 |
| Master metabolic procedure (Get:0044238) | one.88 | nine.00E-ten |
| Metabolic procedure (Go:0008152) | one.72 | ii.16E-08 |
| Cellular process (Become:0009987) | 1.38 | half dozen.49E-03 |
| Unclassified (UNCLASSIFIED) | 0.67 | 0.00E+00 |
Relationship of differentially expressed exosome proteins with leakage/peri-exosomal proteins. (a) Volcano plot for proteins differentially increased in 4°C storage supernatant vs. the original launder. Y centrality, – log10 transformed p value of two samples; the 10 axis is the fold change of protein abundance. (b) Volcano plot for proteins differentially increased in −lxxx°C storage supernatant vs. the launder.
Conversely, 194 proteins were significantly increased in the exosome supernatant afterward −eighty°C storage relative to the starting launder buffer (Figure 8(b), Supplemental Table S5). The major biological processes encoded past these proteins were "chromatin organization" and "protein folding" (Table five), functions as well distinct from those of the parent exosome. We note that the affluence of the proteins increased in the supernatants in either status was greater than the number of proteins depleted from the exosomes during storage (cf. Figure vi(c)).
Tabular array v.
Biological processes of leak/peri-exosomal proteins enriched at −eighty°C storage. A total of 194 loftier-confidence proteins enriched in the supernatant after −80°C storage were analysed for biological processes by GO-Slim (Panther database). Shown are the fold enrichment of the pathway and the p-value (Bonferroni correction).
| PANTHER GO-Slim biological procedure | Enrichment | p- value |
|---|---|---|
| Chromatin organization (Become:0006325) | x.58 | 1.91E-fourteen |
| Poly peptide folding (GO:0006457) | 8.62 | 1.27E-03 |
| Protein complex assembly (GO:0006461) | 8.half dozen | 2.18E-05 |
| Protein circuitous biogenesis (Go:0070271) | 8.55 | 2.31E-05 |
| Organelle system (Become:0006996) | 4.68 | iii.26E-09 |
| Cellular component biogenesis (Go:0044085) | 4.18 | iv.50E-04 |
| Proteolysis (GO:0006508) | three.88 | 1.44E-04 |
| Cellular component organization or biogenesis (GO:0071840) | 3.41 | i.23E-11 |
| Cellular component arrangement (Become:0016043) | 3.15 | 1.61E-08 |
| Catabolic process (GO:0009056) | 2.79 | 3.64E-02 |
| Protein metabolic process (Become:0019538) | ii.49 | v.96E-06 |
| Chief metabolic process (Go:0044238) | 1.84 | 6.66E-08 |
| Metabolic process (Become:0008152) | one.68 | i.39E-06 |
| Unclassified (UNCLASSIFIED) | 0.68 | 0.00E+00 |
| Response to stimulus (Become:0050896) | 0.38 | iii.02E-02 |
Give-and-take
Exosomes have been implicated in the progression of diseases of the airway mucosa, including asthma and chronic obstructive lung diseases.[25,26,42,43] In addition, we have recently demonstrated that exosome secretion is a significant component of the epithelial innate immune response.[18] Understanding how airway epithelial exosomes influence viral induced inflammation and remodelling volition be dependent on isolation conditions that preserve exosomes in biologically agile and native states. Here we report our studies on the systematic effects of storage on airway exosome characteristics and content. Prepared by a standard ultracentrifugation technique, our exosome preparations meet the International Club for Extracellular Vesicles (ISEV) definitions of exosome-enriched fractions [44] by their isolation from an extracellular fluid (BALF), composition as membrane-bound vesicles, size distribution and enrichment of established exosome markers by comparing to the ExoCarta database and Western absorb (see Figure 2, Supplemental Figure 1, Tables 1 and ii). Using this exosome-enriched preparation, our study indicates that different methods of storage accept pregnant furnishings on exosome construction, surface characteristics and protein content. Our findings too identify the presence of storage-labile external proteins coating airway exosomes, proteins we refer to as "peri-exosomal". We will discuss potential mechanisms for these storage phenomena and their implications for exosome diagnostics.
The most significant finding of our study is that freezing airway exosomes results in increased size, promoting multilamellar vesicle formation and aggregation. These findings are consistent with others that accept observed size changes in exosomes subjected to freeze/thaw treatment.[45] As membrane-coated vesicles, we contend that studies on liposome behaviour are relevant to exosomal behaviour. Previous piece of work showed that freezing liposomes induces the formation of multilamellar vesicles.[40,41] Freezing induces multilamellar vesicles by a process involving expansion of ice nano- or micro-crystals in the lipid bilayer and consequent membrane disruption.[46] These microcrystals produce de-mixing of biological surfactants, resulting in fusion betwixt miscible membranes.[47,48] These phenomena are clearly reflected in the morphological changes of frozen exosomes observed in our report (Effigy 2(c) and Supplementary Figure 2). Structural disruption and fusion of exosomes has meaning impact on advancing airway diagnostics. For example, our studies discovered that exosome content varies by epithelial cell type.[xviii] Fusion and subsequent mixing of biological molecules produced by freezing and thawing will have profound consequences for the ability of microfluidic [49] and magnetic-bead based technologies [50] to monitor these individual exosome populations. The add-on of trehalose has recently been suggested to block the formation of ice nano- micro-crystals.[51] All the same, this trouble has not been solved because strategies for avoiding the de-mixing phenomena have nonetheless to be developed.
Another finding of our study is that storage atmospheric condition affects the proteomic content of airway exosomes. Analysis of prison cell-culture derived exosomes has shown that storage at +4°C has a significant impact on CD63 and Hsp70 content,[46] a finding validated the relative depletion of CD63 by Western absorb in our stored samples (Supplemental Figure 1). Our unbiased LC-MS/MS studies take significantly extended the spectrum of proteins depleted by storage at +4°C and −lxxx°C. Interestingly to us, distinct populations of proteins are lost under these two storage conditions. These differences map to specific biological functions encoding coagulation, ubiquitin-mediated proteolysis and chemokine inflammatory pathways. Consequently, storage atmospheric condition may have a meaning consequence on biological functions of airway exosomes, a conclusion supported by the findings that one day of storage at either −20°C or +iv°C affected the anti-bacterial effect of neutrophilic exosomes.[47] We are aware that others have found that storage at −80°C prior to exosome isolation does non accept a pregnant effect on poly peptide contents by MS, or change in exosome morphology.[28] In that written report, whole plasma was frozen before exosome isolation, whereas in our written report, storage occurred after purification. One explanation for this discrepancy is that the presence of concentrated plasma proteins could help to minimize the furnishings of freezing on exosomal fusion.
In experiments designed to empathise the furnishings of storage conditions on exosome protein leakage, nosotros identified over 224 high-confidence proteins that announced in the storage supernatant that were not initially present in the initial launder. These may represent proteins that are initially tightly associated with the exosomes that later on dissociate, or proteins that are secreted from the exosome, or those that may exist released from lysis. Considering the biological functions of the storage supernatant proteins are singled-out from that of the exosome population, we think it unlikely that exosome lysis is the caption. Similarly, nosotros know of no studies that accept demonstrated that exosomes secrete proteins actively. Rather, we translate the appearance of proteins in storage solutions every bit most likely due to dissociation of not-plasma membrane-integrated "peri-exosomal proteins". Exosome preparations isolated from complex biological fluids are coated with proteins in the biological fluids from which they are found. For example, exosomes isolated from human ciliated tracheal bronchial epithelial cells are associated with filamentous mucins (MUCs) 1 and 4, proteins uniquely found within the airway fluid.[25,39] Past contrast, exosomes isolated from plasma are bound by serum proteins, including albumin.[48] Because exosomal accuse density is influenced by the presence of surface-associated proteins, this phenomenon may exist explained by our finding that 194 proteins dissociate from the frozen exosomes. Since exosomes are idea to mediate intercellular signal transduction by straight fusion or endosomal uptake past target cells,[1,2] we call up our findings accept of import implications for the experimental design of studies to understand how airway exosomes participate in prison cell–cell communication. These peri-exosomal proteins may influence exosome targeting and biological behaviour in ways that are not presently fully understood.
In conclusion, we believe these findings are important for informing approaches for functional studies and biosensing assays of airway exosomes. Specifically, the freeze–thaw cycle leads to drastic changes in biophysical properties, content and biomarkers present on the external surfaces of exosomes. Our studies provide critical information for how airway exosomes tin be stored to best preserve their biological function and content, enabling their utilize as biomarkers and/or therapeutic targets.
Supplementary Cloth
Acknowledgements
Core laboratory back up was provided by the Sealy Center for Molecular Medicine Selected Reaction Monitoring facility and the UTMB Optical imaging and Histochemistry Facilities. We give thanks Dr David Konkel for editing the manuscript.
Biography
•
RM, YZ, MJ, HW, MK, YZ, JL, HS performed and interpreted experiments. YZ, CC, SB, MM and ARB conceived and designed experiments. ARB, RM, YZ, MJ, SB, and MM wrote the manuscript. All authors approved the concluding submitted manuscript.
Funding Statement
Research support was provided by the Sealy Center for Molecular Medicine, NIAID Signaling in Airway inflammation PO1 AI068865 (ARB), UTMB CTSA UL1TR001439 (ARB), NIEHS P30 ES006676 (ARB), and DMS-1361411/DMS-1361318 (ARB). SHB and CTC acknowledge back up from the Johnson Cancer Center at Kansas State University.
Disclosure statement
No potential conflict of interest was reported by the authors.
Supplemental data
Supplemental data for this article tin can exist accessed here.
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556670/
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