This study was supported by a research grant from National Institutes of Health grants (NIH) R01HL147095, R01HL141917, and R01HL136431, Kowa Company, Ltd., and the European Union&#39;s Horizon 2020 Research and Innovation Program under the Marie Sk&#322;odowska-Curie Grant Agreement No. 101023041 (R. Cahalane). Figure 1 was created with Biorender.com. The current cleaning protocol was developed by modifying a recommended tube cleaning protocol presented at the International Society of Extracellular Vesicles 2023 Education Day (https://www.youtube.com/watch?v=DOebcOes6iI). Many thanks to Dr. Kathryn Howe and Dr. Sneha Raju from the University Health Network (University of Toronto, Canada) for the original carotid tissue EV samples.

1. Tube cleaning
NOTE: The EV isolation procedure uses both capped and uncapped polycarbonate UC tubes (detailed below). The same procedure was followed for both capped and uncapped tubes. In the case of capped tubes, the lid parts were cleaned individually and reassembled post drying and pre-storage.
<ol>
	<li>Following the initial use of the polycarbonate tubes and removal of the sample, immediately submerge UC tubes in tap water to prevent drying of the sample to the side of the tube.</li>
	<li>Transfer and submerge tubes in 0.1% sodium dodecyl sulfate (SDS) in tap water for 10 min.</li>
	<li>One tube at a time, decant most, but not all, of the SDS solution from the tube and use a cotton swab to thoroughly wipe the area of the tube where the EV pellet was located. 
	NOTE: Do not use anything with bristles. This protocol used normal cotton swabs; however, specialized, low-lint cotton swabs can be used for cleaning the tubes.</li>
	<li>Rinse at least 3x with hot tap water (~50 &#176;C). Make sure to fill and decant the tube completely.</li>
	<li>Rinse 1x with demineralized water using a spray bottle or a narrow-mouthed wash bottle.</li>
	<li>Rinse 1x with 70% ethanol using a spray bottle.</li>
	<li>Leave the tube upside down to dry. 
	NOTE: Tube racks for fluorescence activated cell sorting work well for drying the UC tubes.</li>
	<li>Place the completely dried tubes in a clean jar; they are ready for reuse. 
	NOTE: Currently, the protocol is validated for re-use up to 2x.</li>
</ol>
2. EV enrichment
NOTE: The following EV isolation and proteomics protocol was used for both the original tissue EV isolation, as well as the &#34;mock samples&#34; used to obtain the blank (never-used) and cleaned tube samples. The original samples were resuspended tissue-entrapped EVs from carotid plaques of patients who underwent carotid endarterectomies. Surgical specimens were collected from the University Health Network (Toronto, Canada) and was approved by the institutional ethics committee. All patients provided informed consent for sample collection and data analysis, in accordance with the Declaration of Helsinki. The mock samples were pseudo-pellets obtained from treating blank and cleaned tubes with the same solutions and processing steps as the EV isolation samples.
<ol>
	<li>Defrost EV samples or mock samples in PBS on ice.</li>
	<li>Make NTE buffer (10 mM Tris-HCl, 1 mM EDTA, 0.137 M NaCl pH 7.4) and sterile filter.</li>
	<li>Spin the samples at 10,000 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Move the supernatant into a 1 mL open-top polycarbonate tube.</li>
	<li>Centrifuge the supernatant at 100,000&#215; g for 1 h at 4 &#176;C.</li>
	<li>Resuspend the pellet in 150 &#181;L of sterile-filtered NTE buffer with protease inhibitor.</li>
	<li>Transfer to a 1.5 mL microcentrifuge tube and add NTE buffer to reach a total sample volume of 1.5 mL.</li>
	<li>Keep the samples on ice.</li>
</ol>
3. Preparation of density gradients and density gradient separation
NOTE: This EV isolation protocol has been previously described by Blaser et al.9 In brief:
<ol>
	<li>Prepare five solutions of Iodixanol Density Gradient Medium (10%, 15%, 20%, 25%, and 30%) with NTE buffer as a diluent.</li>
	<li>Layer the five density solutions sequentially in capped ultracentrifuge tube(s) with 30% at the bottom and 10% on the top.</li>
	<li>Secure the caps on the tube(s) and move gently to a stable horizontal position for 1 h at room temperature.</li>
	<li>After 1 h, move the tube(s) gently to a vertical position on ice for 30 min to cool the gradient.</li>
	<li>Add 1.5 mL of the sample or mock sample (NTE buffer) to the top of the prepared density gradient tube(s).</li>
	<li>Ultracentrifuge tube(s) at 250,000 &#215; g for 40 min at 4 &#176;C.</li>
	<li>Remove the top fractions of the density gradient (2.4 mL) to uncapped ultracentrifuge tube(s). Fill to 9 mL with sterile-filtered NTE buffer.</li>
	<li>Pellet the EVs by ultracentrifuging at 100,000 &#215; g for 1 h at 4 &#176;C. 
	NOTE: Circle the location of the visible EV pellet or expected location with a marker. This will allow you to clean the area of the pellet with the swab directly.</li>
	<li>Aspirate the supernatant while moving the tube into an upside-down position for 3 min. Remove the remaining droplets with a cotton swab before turning the tube right side up.</li>
	<li>Resuspend the EV pellet in 12 &#181;L of lyse buffer in the referenced kit.</li>
</ol>
4. Peptide preparation for proteomics
NOTE: Proteomics sample preparation was performed according to the kit protocol with in-house modifications for low-abundance protein samples. The modified protocol is as follows:
<ol>
	<li>Lyse, denature, reduce, and alkylate the samples by heating in lyse buffer at 95 &#176;C for 10 min.</li>
	<li>Digest the samples using the Lys-C/trypsin digest solution for 2 h at 37 &#176;C using 10 &#181;L of lyse solution from the sample and 10 &#181;L of the digest solution.</li>
	<li>Purify peptides through a cartridge by washing hydrophilic and hydrophobic contaminants out and eluting off purified peptides using kit solutions.</li>
	<li>Dry the purified peptides in a speed vacuum at 45 &#176;C for 45 min or until dry. 
	NOTE: Avoid overdrying of the peptides.</li>
	<li>Store samples at -80 &#176;C until the time of mass spectrometry sequencing.</li>
	<li>Add 17 &#181;L of LC-load solution to the dried peptides.</li>
	<li>Leave the samples on ice for 1 h.</li>
	<li>Vortex the samples briefly and then sonicate in an ultrasonic waterbath for 5 min.</li>
	<li>Centrifuge the LC-load samples at 16,000 &#215; g for 1 min, aspirate 15 &#181;L from the top, and separate into a new tube.</li>
</ol>
5. Liquid chromatography and tandem mass spectrometry sequencing
<ol>
	<li>Inject 2 &#181;L of LC-load peptide solution onto a mass spectrometer.</li>
	<li>Fractionate the peptides using a dual-column setup.</li>
	<li>Heat the column at a constant temperature of 45 &#730;C.</li>
	<li>Run the analytical gradient at 300 nL/min from 5% to 21% Solvent B (acetonitrile/0.1% formic acid) for 60 min, followed by 10 min of 21% to 30% Solvent B, and another 10 min of a 95%-5% jigsaw wash.</li>
	<li>Set the MS1 to 120 K resolution (maximum injection time, 25 ms), subject the top precursor ions (within a 3.0 s cycle time) to HCD isolation width 1.2 m/z, dynamic exclusion enabled (60 s), and set the MS2 resolution to 60 K (maximum injection time, auto; normalized collision energy, 24%, 26%, and 28%).</li>
	<li>Set the MS1 and MS2 acquisition range to 400 m/z-1,200 m/z and 120 m/z-1,200 m/z, respectively.</li>
</ol>
6. Peptide/protein identification
<ol>
	<li>Search the acquired peptide spectra with a proteomic search software&#160;using a commercial search algorithm against a human uniProt database.</li>
	<li>Set the digestion enzyme to trypsin and allow up to two missed cleavages.</li>
	<li>Set the precursor tolerance to 10 ppm and the fragment tolerance window to 0.02 Da.</li>
	<li>Set methionine oxidation and N-terminal acetylation as variable modifications and cysteine carbamidomethylation as fixed modification.</li>
	<li>Filter the peptides based on a false discovery rate (FDR) threshold of 1.0% as calculated by the Percolator algorithm.</li>
	<li>Only consider peptides assigned to one given protein group and not detected in any other protein group as unique and use them for further analyses.</li>
	<li>Only include a minimum of two unique peptides for each protein in the analyses.</li>
	<li>Maximize IDs with precursor ion detection.</li>
	<li>Perform chromatographic alignment with a maximum retention time shift of 10 min, mass tolerance of 10 ppm, and signal-to-noise minimum of 5.</li>
	<li>Normalize peptide abundance by total peptide amount.</li>
</ol>

Efficient Recycling of Polycarbonate Ultracentrifuge Tubes for Proteomics

<ol>
	<li>van Niel, G., D'Angelo, G., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shedding+light+on+the+cell+biology+of+extracellular+vesicles.">Shedding light on the cell biology of extracellular vesicles.</a> Nature Reviews Molecular Cell Biology. 19 (4), 213-228 (2018).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Witwer, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> Journal of Extracellular Vesicles. 2 (1), 20360 (2013).</li><li>Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+extracellular+vesicles:+general+methodologies+and+latest+trends.">Isolation of extracellular vesicles: general methodologies and latest trends.</a> BioMed Research International. 2018, e854347 (2018).</li><li>Kowal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proceedings of the National Academy of Sciences. 113 (8), E968-E977 (2016).</li><li>Karimi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+analysis+of+the+plasma+extracellular+vesicle+proteome+after+separation+from+lipoproteins.">Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.</a> Cellular and Molecular Life Sciences. 75 (15), 2873-2886 (2018).</li><li>Lischnig, A., Bergqvist, M., Ochiya, T., L&#228;sser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+identifies+proteins+enriched+in+large+and+small+extracellular+vesicles.">Quantitative proteomics identifies proteins enriched in large and small extracellular vesicles.</a> Molecular &amp; Cellular Proteomics. 21 (9), 100273 (2022).</li><li>van Herwijnen, M. J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+proteomic+analysis+of+human+milk-derived+extracellular+vesicles+unveils+a+novel+functional+proteome+distinct+from+other+milk+components.">Comprehensive proteomic analysis of human milk-derived extracellular vesicles unveils a novel functional proteome distinct from other milk components.</a> Molecular &amp; Cellular Proteomics. 15 (11), 3412-3423 (2016).</li><li>Blaser, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiomics+of+tissue+extracellular+vesicles+identifies+unique+modulators+of+atherosclerosis+and+calcific+aortic+valve+stenosis.">Multiomics of tissue extracellular vesicles identifies unique modulators of atherosclerosis and calcific aortic valve stenosis.</a> Circulation. 148 (8), 661-678 (2023).</li><li>Kristensen, K., Henriksen, J. R., Andresen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+cationic+peptides+to+solid+surfaces+of+glass+and+plastic.">Adsorption of cationic peptides to solid surfaces of glass and plastic.</a> PLOS ONE. 10 (5), e0122419 (2015).</li><li>Use and care of centrifuge tubes and bottles. Beckman Coulter Available from: <a target="_blank" href="https://www.beckman.com/supplies/tubes-and-bottles">https://www.beckman.com/supplies/tubes-and-bottles</a> (2014)</li><li>Wang, L., Zhang, J., Hou, S., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+quantifying+polycarbonate+and+polyethylene+terephthalate+microplastics+in+environmental+samples+by+liquid+chromatography-tandem+mass+spectrometry.">A simple method for quantifying polycarbonate and polyethylene terephthalate microplastics in environmental samples by liquid chromatography-tandem mass spectrometry.</a> Environmental Science &amp; Technology Letters. 4 (12), 530-534 (2017).</li><li>Schittek, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermcidin:+a+novel+human+antibiotic+peptide+secreted+by+sweat+glands.">Dermcidin: a novel human antibiotic peptide secreted by sweat glands.</a> Nature Immunology. 2 (12), 1133-1137 (2001).</li><li>Blaydon, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+CSTA,+encoding+Cystatin+A,+underlie+exfoliative+ichthyosis+and+reveal+a+role+for+this+protease+inhibitor+in+cell-cell+adhesion.">Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion.</a> American Journal of Human Genetics. 89 (4), 564-571 (2011).</li><li>Henry, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hornerin+is+a+component+of+the+epidermal+cornified+cell+envelopes.">Hornerin is a component of the epidermal cornified cell envelopes.</a> FASEB Journal. 25 (5), 1567-1576 (2011).</li><li>Bolling, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+ichthyotic+peeling+skin+syndrome+due+to+FLG2+mutations.">Generalized ichthyotic peeling skin syndrome due to FLG2 mutations.</a> The Journal of Investigative Dermatology. 138 (8), 1881-1884 (2018).</li><li>Jin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAMP+molecules+S100A9+and+S100A8+activated+by+IL-17A+and+house-dust+mites+are+increased+in+atopic+dermatitis.">DAMP molecules S100A9 and S100A8 activated by IL-17A and house-dust mites are increased in atopic dermatitis.</a> Experimental Dermatology. 23 (12), 938-941 (2014).</li><li>Gl&#228;ser, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+protein+psoriasin+(S100A7)+is+upregulated+in+atopic+dermatitis+and+after+experimental+skin+barrier+disruption.">The antimicrobial protein psoriasin (S100A7) is upregulated in atopic dermatitis and after experimental skin barrier disruption.</a> Journal of Investigative Dermatology. 129 (3), 641-649 (2009).</li><li>Molhave, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=House+dust+in+seven+Danish+offices.">House dust in seven Danish offices.</a> Atmospheric Environment. 34, 4767-4779 (1999).</li><li>Buschow, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-associated+proteins+in+B-cell+exosomes+and+potential+functional+implications+for+exosome+biogenesis.">MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis.</a> Immunology and Cell Biology. 88 (8), 851-856 (2010).</li></ol>

The authors have no conflicts of interest to disclose.

Here we describe and validate a protocol for cleaning polycarbonate UC tubes for EV enrichment and proteomic applications. We demonstrated the successful removal of residual protein from the previous UC tube sample compared with a cleaned pseudo-pellet analysis below the limit of detection of this mass spectrometry acquisition protocol and showed the proteomic similarity of blank never-used UC tube compared to cleaned UC tube pseudo pellets.
First, to prevent the inadvertent adsorption of prot...

Extracellular vesicles (EVs) are lipid-bilayer-delimited particles released from cells that carry biologically active cargo, such as protein, and participate in various biological processes, including cell-cell communication and the formation of biologic mineralization1. These particles are found in all body fluids and tissues, and their biological activities and uses are a rapidly evolving field of scientific research. Isolation and validation of these nanoparticles present various challenges due to their small size and bio-similarity to other particles, such as liposomes and protein aggregates. The most recent International Society of Extracellular Vesicles guidelines, Minimal information for studies of extracellular vesicles 2018 (MISEV2018), are the recognized standard for EV scientific research2.
Various methods, often in tandem, must be used for EV isolation depending on the upstream source and the downstream applications. As of 2015, the most common primary isolation method for EVs was differential ultracentrifugation (UC)2. In principle, initial lower speed centrifugation separates&#160;larger or denser unwanted components, such as cells and cell debris, leaving EVs in the supernatant. Subsequently, UC uses very high centrifugal force to pellet out and thus separate and purify EVs from other particles that are smaller or less dense but which may also contain dense non-EV particles. Most protocols will often use, at one step or another, UC to isolate EVs from a fluid3. Further, UC is used in other methods of EV isolation, such as density gradient ultracentrifugation (DGUC), which uses mediums such as OptiprepTM iodixanol and centrifugal force to separate EVs according to their buoyant density4. Other methods of EV isolation exist3.
Considering the rapidly evolving understanding of the biological processes dictated by EVs and their potential as biomarkers and information regarding pathophysiology, discovery-based analyses such as proteomics have gained traction5,6,7,8. EVs are small, and, depending on the source, have a low yield of protein (&#60;1 &#956;g) when compared to whole tissue or cell lysate. Isolation of EVs for proteomics analysis requires special considerations, such as the removal of non-EV protein contaminants from upstream liquid or tissue, consideration of EV protein degradation during the isolation process, and the utilization of methods that create protein solutions that are chemically compatible with peptide preparation and mass spectrometry analyses.
Research laboratory consumables are often plastic and disposable in nature. These single-use materials contribute to the global plastic pollution crisis and consumable costs. Specialized polycarbonate and polystyrene UC tubes are designed to withstand the high centrifugal forces required to pellet EVs in laboratory applications. While it is possible to sterilize and disinfect UC tubes for re-use, proteomic analysis, especially those of low protein yields such as EVs, requires special attention. Not only is sufficient removal of residual protein from the previous use paramount, chemical compatibility with mass-spectroscopy and plastic-derived polymer contamination must also be considered.
Here we present a cleaning protocol of polycarbonate tubes using detergents suitable for mass-spectrometry and perform experiments to validate its successful removal of residual protein below limits of detection and lack of detectable polymer contaminants. To validate the cleaning protocol for EV proteomic applications using both UC and DGUC purposes, we obtained tubes from isolations of human vasculature tissue EVs with a combined UC and DGUC protocol. Tubes were cleaned using the protocol described, and the experimental process was repeated without samples comparing a blank never-used UC tube and a cleaned UC tube. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the cost associated with such experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Open-Top Thickwall Polycarbonate Tube</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>355630</td>
			<td>uncapped ultracentrifuge tube(s)&#160;</td>
		</tr>
		<tr>
			<td>10.4 mL Polycarbonate Bottle with Cap Assembly</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>355603</td>
			<td>capped ultracentrifuge tube(s)&#160;</td>
		</tr>
		<tr>
			<td>an Acclaim PepMap 100 C18 HPLC Columns, 75 &#181;m x 70 mm; and an EASY-Spray HPLC Column, 75 &#181;m x 250 mm</td>
			<td>ThermoFisher Scientific</td>
			<td>164946 and ES902</td>
			<td>Dual column setup</td>
		</tr>
		<tr>
			<td>Critical Swab Swab, Cotton Head</td>
			<td>VWR</td>
			<td>89031-270</td>
			<td>cotton swab</td>
		</tr>
		<tr>
			<td>Exploris 480 fronted with EASY-Spray Source, coupled to an Easy-nLC1200 HPLC pump. &#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>BRE725533</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr>
			<td>Human UniProt database (101043 entries, updated January 2022)</td>
			<td>NA</td>
			<td>NA</td>
			<td>Human database</td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>water</td>
		</tr>
		<tr>
			<td>PreOmics iST kit &#160;</td>
			<td>PreOmics</td>
			<td>P.O.00027</td>
			<td>commercial protein sample preparation kit&#160;</td>
		</tr>
		<tr>
			<td>Proteome Discoverer &#160;package (PD, Version 2.5)</td>
			<td>ThermoFisher Scientific</td>
			<td>NA</td>
			<td>Proteomic search software</td>
		</tr>
		<tr>
			<td>SEQUEST-HT search algorithm&#160;</td>
			<td>NA</td>
			<td>NA</td>
			<td>Search algorithm</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (20%)</td>
			<td>Boston BioProducts</td>
			<td>BM-230</td>
			<td>detergent</td>
		</tr>
	</tbody>
</table>

polycarbonate ultracentrifuge tube re-use in proteomic analyses of extracellular vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins.
This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples.
Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (&#8804;20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

To validate the cleaning protocol (Figure 1), two experiments were performed. First, the proteome of the &#34;mock sample&#34; from the cleaned tube was compared against the proteome of the tissue EV sample from the tube&#39;s initial use to determine the carryover of identified proteins. Representative chromatograms show a reduction in peak heterogeneity following cleaning of the tubes (Figure 2). In the original EV isolation, 806 proteins were identified with ...

Read this Scientific Article Protocol about Author Spotlight: Effective Reuse of Polycarbonate Tubes for Extracellular Vesicle Isolation at JoVE.com

Author Spotlight: Effective Reuse of Polycarbonate Tubes for Extracellular Vesicle Isolation

A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments.

Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ...

polycarbonate-ultracentrifuge-tube-re-use-proteomic-analyses

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Biology

806 Views.  Brigham and Women’s Hospital, Harvard Medical School. A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments.

Video: Author Spotlight: Effective Reuse of Polycarbonate Tubes for Extracellular Vesicle Isolation

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...