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Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis

BACKGROUND: Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. Furthermo...

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Autores principales: Guldbrandsen, Astrid, Lereim, Ragnhild Reehorst, Jacobsen, Mari, Garberg, Hilde, Kroksveen, Ann Cathrine, Barsnes, Harald, Berven, Frode S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499868/
https://www.ncbi.nlm.nih.gov/pubmed/32963504
http://dx.doi.org/10.1186/s12014-020-09296-5
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author Guldbrandsen, Astrid
Lereim, Ragnhild Reehorst
Jacobsen, Mari
Garberg, Hilde
Kroksveen, Ann Cathrine
Barsnes, Harald
Berven, Frode S.
author_facet Guldbrandsen, Astrid
Lereim, Ragnhild Reehorst
Jacobsen, Mari
Garberg, Hilde
Kroksveen, Ann Cathrine
Barsnes, Harald
Berven, Frode S.
author_sort Guldbrandsen, Astrid
collection PubMed
description BACKGROUND: Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. Furthermore, verification studies are often based on a low number of proteins from a single discovery experiment in medium-sized cohorts, where antibodies and surrogate peptides may differ, thus only providing an indication of proteins affected by the disease and not revealing the bigger picture or concluding on the validity of the markers. We here present a standard approach for locating promising biomarker candidates based on existing knowledge, resulting in high-quality assays covering the main biological processes affected by multiple sclerosis for comparable measurements over time. METHODS: Biomarker candidates were located in CSF-PR (proteomics.uib.no/csf-pr), and further filtered based on estimated concentration in CSF and biological function. Peptide surrogates for internal standards were selected according to relevant criteria, parallel reaction monitoring (PRM) assays created, and extensive assay quality testing performed, i.e. intra- and inter-day variation, trypsin digestion status over time, and whether the peptides were able to separate multiple sclerosis patients and controls. RESULTS: Assays were developed for 25 proteins, represented by 72 peptides selected according to relevant guidelines and available literature and tested for assay peptide suitability. Stability testing revealed 64 peptides with low intra- and inter-day variations, with 44 also being stably digested after 16 h of trypsin digestion, and 37 furthermore showing a significant difference between multiple sclerosis and controls, thereby confirming literature findings. Calibration curves and the linear area of measurement have, so far, been determined for 17 of these peptides. CONCLUSIONS: We present 37 high-quality PRM assays across 21 CSF-proteins found to be affected by multiple sclerosis, along with a recommended workflow for future development of new assays. The assays can directly be used by others, thus enabling better comparison between studies. Finally, the assays can robustly and stably monitor biological processes in multiple sclerosis patients over time, thus potentially aiding in diagnosis and prognosis, and ultimately in treatment decisions.
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spelling pubmed-74998682020-09-21 Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis Guldbrandsen, Astrid Lereim, Ragnhild Reehorst Jacobsen, Mari Garberg, Hilde Kroksveen, Ann Cathrine Barsnes, Harald Berven, Frode S. Clin Proteomics Research BACKGROUND: Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. Furthermore, verification studies are often based on a low number of proteins from a single discovery experiment in medium-sized cohorts, where antibodies and surrogate peptides may differ, thus only providing an indication of proteins affected by the disease and not revealing the bigger picture or concluding on the validity of the markers. We here present a standard approach for locating promising biomarker candidates based on existing knowledge, resulting in high-quality assays covering the main biological processes affected by multiple sclerosis for comparable measurements over time. METHODS: Biomarker candidates were located in CSF-PR (proteomics.uib.no/csf-pr), and further filtered based on estimated concentration in CSF and biological function. Peptide surrogates for internal standards were selected according to relevant criteria, parallel reaction monitoring (PRM) assays created, and extensive assay quality testing performed, i.e. intra- and inter-day variation, trypsin digestion status over time, and whether the peptides were able to separate multiple sclerosis patients and controls. RESULTS: Assays were developed for 25 proteins, represented by 72 peptides selected according to relevant guidelines and available literature and tested for assay peptide suitability. Stability testing revealed 64 peptides with low intra- and inter-day variations, with 44 also being stably digested after 16 h of trypsin digestion, and 37 furthermore showing a significant difference between multiple sclerosis and controls, thereby confirming literature findings. Calibration curves and the linear area of measurement have, so far, been determined for 17 of these peptides. CONCLUSIONS: We present 37 high-quality PRM assays across 21 CSF-proteins found to be affected by multiple sclerosis, along with a recommended workflow for future development of new assays. The assays can directly be used by others, thus enabling better comparison between studies. Finally, the assays can robustly and stably monitor biological processes in multiple sclerosis patients over time, thus potentially aiding in diagnosis and prognosis, and ultimately in treatment decisions. BioMed Central 2020-09-18 /pmc/articles/PMC7499868/ /pubmed/32963504 http://dx.doi.org/10.1186/s12014-020-09296-5 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Guldbrandsen, Astrid
Lereim, Ragnhild Reehorst
Jacobsen, Mari
Garberg, Hilde
Kroksveen, Ann Cathrine
Barsnes, Harald
Berven, Frode S.
Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
title Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
title_full Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
title_fullStr Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
title_full_unstemmed Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
title_short Development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
title_sort development of robust targeted proteomics assays for cerebrospinal fluid biomarkers in multiple sclerosis
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499868/
https://www.ncbi.nlm.nih.gov/pubmed/32963504
http://dx.doi.org/10.1186/s12014-020-09296-5
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