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Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma
Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10094439/ https://www.ncbi.nlm.nih.gov/pubmed/37047281 http://dx.doi.org/10.3390/ijms24076290 |
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author | Templeton, Evelyn M. Pilbrow, Anna P. Kleffmann, Torsten Pickering, John W. Rademaker, Miriam T. Scott, Nicola J. A. Ellmers, Leigh J. Charles, Christopher J. Endre, Zoltan H. Richards, A. Mark Cameron, Vicky A. Lassé, Moritz |
author_facet | Templeton, Evelyn M. Pilbrow, Anna P. Kleffmann, Torsten Pickering, John W. Rademaker, Miriam T. Scott, Nicola J. A. Ellmers, Leigh J. Charles, Christopher J. Endre, Zoltan H. Richards, A. Mark Cameron, Vicky A. Lassé, Moritz |
author_sort | Templeton, Evelyn M. |
collection | PubMed |
description | Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R(2) = 0.85–0.90) and plasma (SPEED R(2) = 0.84; SDC R(2) = 0.76, S-Trap R(2) = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired. |
format | Online Article Text |
id | pubmed-10094439 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-100944392023-04-13 Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma Templeton, Evelyn M. Pilbrow, Anna P. Kleffmann, Torsten Pickering, John W. Rademaker, Miriam T. Scott, Nicola J. A. Ellmers, Leigh J. Charles, Christopher J. Endre, Zoltan H. Richards, A. Mark Cameron, Vicky A. Lassé, Moritz Int J Mol Sci Article Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R(2) = 0.85–0.90) and plasma (SPEED R(2) = 0.84; SDC R(2) = 0.76, S-Trap R(2) = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired. MDPI 2023-03-27 /pmc/articles/PMC10094439/ /pubmed/37047281 http://dx.doi.org/10.3390/ijms24076290 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Templeton, Evelyn M. Pilbrow, Anna P. Kleffmann, Torsten Pickering, John W. Rademaker, Miriam T. Scott, Nicola J. A. Ellmers, Leigh J. Charles, Christopher J. Endre, Zoltan H. Richards, A. Mark Cameron, Vicky A. Lassé, Moritz Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma |
title | Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma |
title_full | Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma |
title_fullStr | Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma |
title_full_unstemmed | Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma |
title_short | Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma |
title_sort | comparison of speed, s-trap, and in-solution-based sample preparation methods for mass spectrometry in kidney tissue and plasma |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10094439/ https://www.ncbi.nlm.nih.gov/pubmed/37047281 http://dx.doi.org/10.3390/ijms24076290 |
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