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Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions

PURPOSE: The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient...

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Autores principales: Snashall, Corinna M., Sutton, Chris W., Faro, Letizia Lo, Ceresa, Carlo, Ploeg, Rutger, Shaheed, Sadr ul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10648346/
https://www.ncbi.nlm.nih.gov/pubmed/37968584
http://dx.doi.org/10.1186/s12014-023-09440-x
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author Snashall, Corinna M.
Sutton, Chris W.
Faro, Letizia Lo
Ceresa, Carlo
Ploeg, Rutger
Shaheed, Sadr ul
author_facet Snashall, Corinna M.
Sutton, Chris W.
Faro, Letizia Lo
Ceresa, Carlo
Ploeg, Rutger
Shaheed, Sadr ul
author_sort Snashall, Corinna M.
collection PubMed
description PURPOSE: The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate. EXPERIMENTAL DESIGN: Two different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography–mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion. RESULTS: In-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate. CONCLUSIONS: This study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12014-023-09440-x.
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spelling pubmed-106483462023-11-15 Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions Snashall, Corinna M. Sutton, Chris W. Faro, Letizia Lo Ceresa, Carlo Ploeg, Rutger Shaheed, Sadr ul Clin Proteomics Methodology PURPOSE: The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate. EXPERIMENTAL DESIGN: Two different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography–mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion. RESULTS: In-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate. CONCLUSIONS: This study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12014-023-09440-x. BioMed Central 2023-11-15 /pmc/articles/PMC10648346/ /pubmed/37968584 http://dx.doi.org/10.1186/s12014-023-09440-x Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/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 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/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://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.
spellingShingle Methodology
Snashall, Corinna M.
Sutton, Chris W.
Faro, Letizia Lo
Ceresa, Carlo
Ploeg, Rutger
Shaheed, Sadr ul
Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
title Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
title_full Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
title_fullStr Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
title_full_unstemmed Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
title_short Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
title_sort comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10648346/
https://www.ncbi.nlm.nih.gov/pubmed/37968584
http://dx.doi.org/10.1186/s12014-023-09440-x
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