Cargando…
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...
Autores principales: | , , , , , , , , |
---|---|
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 |
_version_ | 1783435683651649536 |
---|---|
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. |
format | Online Article Text |
id | pubmed-6632171 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT saitosuguru theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT hiraoyoshitoshi theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT quaderyalif theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT xubo theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT elguoshyamr theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT fujinakahidehiko theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT komashohei theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT yamamotokeiko theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT yamamototadashi theoptimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT saitosuguru optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT hiraoyoshitoshi optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT quaderyalif optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT xubo optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT elguoshyamr optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT fujinakahidehiko optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT komashohei optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT yamamotokeiko optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics AT yamamototadashi optimizedworkflowforsamplepreparationinlcmsmsbasedurineproteomics |