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. 2021 Aug 23;20:172. doi: 10.1186/s12933-021-01359-7

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Two trypsin digestion protocols, followed by fluorescence microscopy and proteomics of platelet poor plasma (PPP) from healthy individuals, patients with Type 2 Diabetes Mellitus (T2DM), COVID-19 and Long COVID/PASC. (1) Citrated blood was centrifuged to obtain PPP. (2) PPP were treated with trypsin to allow plasma protein digestion. Health PPP and T2DM PPP were fully degraded. COVID-19 and Long COVID/PASC sample formed a undigested pellet deposit at the bottom of the tubes. (3 and 4) For fluorescence microscopy, the supernatants were removed and the remaining 10 µL of supernatant and/or pellet samples were exposed to thioflavin T (ThT) and viewed with fluorescence microscope. Before liquid chromatography-mass spectrometry (LC–MS) based proteomics, supernatants were passed through a C₁₈ solid phase extraction (SPE) device. (5) A second trypsin digestion protocol was followed to (6) degrade the pellet deposit in the COVID-19 and Long COVID/PASC samples. The same method was followed with healthy and T2DM PPP (although these samples did not contain a visible pellet deposit). (7) Double-trypsinized samples from controls, COVID-19 and Long COVID/PASC samples were then studied using proteomics. (Figure created with BioRender.com)