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The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform...

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Autores principales: Saito, Suguru, Hirao, Yoshitoshi, Quadery, Ali F., Xu, Bo, Elguoshy, Amr, Fujinaka, Hidehiko, Koma, Shohei, Yamamoto, Keiko, Yamamoto, Tadashi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632171/
http://dx.doi.org/10.3390/mps2020046
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author Saito, Suguru
Hirao, Yoshitoshi
Quadery, Ali F.
Xu, Bo
Elguoshy, Amr
Fujinaka, Hidehiko
Koma, Shohei
Yamamoto, Keiko
Yamamoto, Tadashi
author_facet Saito, Suguru
Hirao, Yoshitoshi
Quadery, Ali F.
Xu, Bo
Elguoshy, Amr
Fujinaka, Hidehiko
Koma, Shohei
Yamamoto, Keiko
Yamamoto, Tadashi
author_sort Saito, Suguru
collection PubMed
description The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.
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spelling pubmed-66321712019-08-19 The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics Saito, Suguru Hirao, Yoshitoshi Quadery, Ali F. Xu, Bo Elguoshy, Amr Fujinaka, Hidehiko Koma, Shohei Yamamoto, Keiko Yamamoto, Tadashi Methods Protoc Benchmark The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery. MDPI 2019-06-07 /pmc/articles/PMC6632171/ http://dx.doi.org/10.3390/mps2020046 Text en © 2019 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Benchmark
Saito, Suguru
Hirao, Yoshitoshi
Quadery, Ali F.
Xu, Bo
Elguoshy, Amr
Fujinaka, Hidehiko
Koma, Shohei
Yamamoto, Keiko
Yamamoto, Tadashi
The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
title The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
title_full The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
title_fullStr The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
title_full_unstemmed The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
title_short The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
title_sort optimized workflow for sample preparation in lc-ms/ms-based urine proteomics
topic Benchmark
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632171/
http://dx.doi.org/10.3390/mps2020046
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