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

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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.

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

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

We study cardiovascular diseases, specifically the role of extracellular vesicles in the pathological calcification of cardiovascular tissues, such as valves and blood vessels. Researchers in our group have recently defined methods to extract tissue and trapped extracellular vesicles from fibro calcific cardiovascular tissue to determine their proteomic profile. Our group has demonstrated that these extracellular vesicles contain important protein cargo for initiating calcification in disease tissues.The specialized tubes used in the ultracentrifugation step of extracellular vesicle isolation can be cleaned and sterilized for reuse. However, proteomic analysis requires special consideration as any contamination could greatly affect downstream results. Here we validate a protocol for reusing consumables, specifically polycarbonate ultracentrifuge tubes for extracellular vesicle isolation that is suitable for downstream low input proteomics applications.Our protocol is simple and effective, and it will allow researchers to reduce both the consumable costs and the waste typically associated with these isolation protocols.

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

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

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

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Biology

629 Views.  Brigham and Women’s Hospital, Harvard Medical School. We study cardiovascular diseases, specifically the role of extracellular vesicles in the pathological calcification of cardiovascular tissues, such as valves and blood vessels. Researchers in our group have recently defined methods to extract tissue and trapped extracellular vesicles from fibro calcific cardiovascular tissue to determine their proteomic profile. Our group has demonstrated that these extracellular vesicles contain important protein cargo for initiating calcification in disease...