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. 2018 Oct 9;8:15039. doi: 10.1038/s41598-018-33280-7

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© The Author(s) 2018

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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The schematic diagram represents the entire process of ELV-SWATH-MS workflow. Three main steps included; (i) the isolation of urinary extracellular vesicles including exosome-like vesicles (ELVs) and microvesicles (MVs) by differential ultracentrifugation and subsequently concentrating urine proteins (UPs) by ultrafiltration (3-kDa cutoff); (ii) the spectral library generation based on all identified fragment ions, peptides and proteins from data-dependent acquisition (DDA) of 16 protein fractionations of ELVs, MVs and UPs and a manual curation to remove the sources of variability in targeted proteomic analysis; (iii) SWATH analysis using predefined targets in the curated spectral library for targeted data extraction and peptide quantitation from the digital protein records of ELVs, MVs and UPs generated by data-independent acquisition (DIA).