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Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions
Snashall, C.M. Sutton, Chris W. Lo Faro, L. Ceresa, C. Ploeg, R. Shaheed, S.U.
Snashall, C.M.
Sutton, Chris W.
Lo Faro, L.
Ceresa, C.
Ploeg, R.
Shaheed, S.U.
Publication Date
2023-11-15
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Rights
(c) 2023 The Authors. This is an Open Access article distributed under the Creative Commons CC-BY license (https://creativecommons.org/licenses/by/4.0/)
Peer-Reviewed
Yes
Open Access status
openAccess
Accepted for publication
2023-10-25
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snashall_et_al_2023.pdf
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Additional title
Abstract
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.
Version
Published version
Citation
Snashall CM, Sutton CW, Lo Faro L et al (2023) Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions. Clinical Proteomics 20(51).
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Article