Cargando…
A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome
Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to addre...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489773/ https://www.ncbi.nlm.nih.gov/pubmed/28587287 http://dx.doi.org/10.3390/proteomes5020013 |
_version_ | 1783246858865344512 |
---|---|
author | D’Silva, Arlene M. Hyett, Jon A. Coorssen, Jens R. |
author_facet | D’Silva, Arlene M. Hyett, Jon A. Coorssen, Jens R. |
author_sort | D’Silva, Arlene M. |
collection | PubMed |
description | Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3–10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7–20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes. |
format | Online Article Text |
id | pubmed-5489773 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-54897732017-06-30 A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome D’Silva, Arlene M. Hyett, Jon A. Coorssen, Jens R. Proteomes Article Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3–10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7–20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes. MDPI 2017-06-06 /pmc/articles/PMC5489773/ /pubmed/28587287 http://dx.doi.org/10.3390/proteomes5020013 Text en © 2017 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 | Article D’Silva, Arlene M. Hyett, Jon A. Coorssen, Jens R. A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome |
title | A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome |
title_full | A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome |
title_fullStr | A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome |
title_full_unstemmed | A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome |
title_short | A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome |
title_sort | routine ‘top-down’ approach to analysis of the human serum proteome |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489773/ https://www.ncbi.nlm.nih.gov/pubmed/28587287 http://dx.doi.org/10.3390/proteomes5020013 |
work_keys_str_mv | AT dsilvaarlenem aroutinetopdownapproachtoanalysisofthehumanserumproteome AT hyettjona aroutinetopdownapproachtoanalysisofthehumanserumproteome AT coorssenjensr aroutinetopdownapproachtoanalysisofthehumanserumproteome AT dsilvaarlenem routinetopdownapproachtoanalysisofthehumanserumproteome AT hyettjona routinetopdownapproachtoanalysisofthehumanserumproteome AT coorssenjensr routinetopdownapproachtoanalysisofthehumanserumproteome |