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Mass spectrometry quantification of clusterin in the human brain

BACKGROUND: The multifunctional glycoprotein clusterin has been associated with late-onset Alzheimer’s disease (AD). Further investigation to define the role of clusterin in AD phenotypes would be aided by the development of techniques to quantify level, potential post-translational modifications, a...

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Autores principales: Chen, Junjun, Wang, Meiyao, Turko, Illarion V
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3470951/
https://www.ncbi.nlm.nih.gov/pubmed/22906254
http://dx.doi.org/10.1186/1750-1326-7-41
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author Chen, Junjun
Wang, Meiyao
Turko, Illarion V
author_facet Chen, Junjun
Wang, Meiyao
Turko, Illarion V
author_sort Chen, Junjun
collection PubMed
description BACKGROUND: The multifunctional glycoprotein clusterin has been associated with late-onset Alzheimer’s disease (AD). Further investigation to define the role of clusterin in AD phenotypes would be aided by the development of techniques to quantify level, potential post-translational modifications, and isoforms of clusterin. We have developed a quantitative technique based on multiple reaction monitoring (MRM) mass spectrometry to measure clusterin in human postmortem brain tissues. RESULTS: A stable isotope-labeled concatenated peptide (QconCAT) bearing selected peptides from clusterin was expressed with an in vitro translation system and purified. This clusterin QconCAT was validated for use as an internal standard for clusterin quantification using MRM mass spectrometry. Measurements were performed on the human postmortem frontal and temporal cortex from control and severe AD cases. During brain tissues processing, 1% SDS was used in the homogenization buffer to preserve potential post-translational modifications of clusterin. However, MRM quantifications in the brain did not suggest phosphorylation of Thr(393), Ser(394), and Ser(396) residues reported for clusterin in serum. MRM quantifications in the frontal cortex demonstrated significantly higher (P < 0.01) level of clusterin in severe AD group (39.1 ± 9.1 pmol/mg tissue protein) in comparison to control group (25.4 ± 4.4 pmol/mg tissue protein). In the temporal cortex, the clusterin levels were not significantly different, 29.0 ± 7.9 pmol/mg tissue protein and 28.0 ± 8.4 pmol/mg tissue protein in control and severe AD groups, respectively. CONCLUSIONS: The proposed protocol is a universal quantitative technique to assess expression level of clusterin. It is expected that application of this protocol to quantification of various clusterin isoforms and potential post-translational modifications will be helpful in addressing the role of clusterin in AD.
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spelling pubmed-34709512012-10-18 Mass spectrometry quantification of clusterin in the human brain Chen, Junjun Wang, Meiyao Turko, Illarion V Mol Neurodegener Methodology BACKGROUND: The multifunctional glycoprotein clusterin has been associated with late-onset Alzheimer’s disease (AD). Further investigation to define the role of clusterin in AD phenotypes would be aided by the development of techniques to quantify level, potential post-translational modifications, and isoforms of clusterin. We have developed a quantitative technique based on multiple reaction monitoring (MRM) mass spectrometry to measure clusterin in human postmortem brain tissues. RESULTS: A stable isotope-labeled concatenated peptide (QconCAT) bearing selected peptides from clusterin was expressed with an in vitro translation system and purified. This clusterin QconCAT was validated for use as an internal standard for clusterin quantification using MRM mass spectrometry. Measurements were performed on the human postmortem frontal and temporal cortex from control and severe AD cases. During brain tissues processing, 1% SDS was used in the homogenization buffer to preserve potential post-translational modifications of clusterin. However, MRM quantifications in the brain did not suggest phosphorylation of Thr(393), Ser(394), and Ser(396) residues reported for clusterin in serum. MRM quantifications in the frontal cortex demonstrated significantly higher (P < 0.01) level of clusterin in severe AD group (39.1 ± 9.1 pmol/mg tissue protein) in comparison to control group (25.4 ± 4.4 pmol/mg tissue protein). In the temporal cortex, the clusterin levels were not significantly different, 29.0 ± 7.9 pmol/mg tissue protein and 28.0 ± 8.4 pmol/mg tissue protein in control and severe AD groups, respectively. CONCLUSIONS: The proposed protocol is a universal quantitative technique to assess expression level of clusterin. It is expected that application of this protocol to quantification of various clusterin isoforms and potential post-translational modifications will be helpful in addressing the role of clusterin in AD. BioMed Central 2012-08-20 /pmc/articles/PMC3470951/ /pubmed/22906254 http://dx.doi.org/10.1186/1750-1326-7-41 Text en Copyright ©2012 Chen et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology
Chen, Junjun
Wang, Meiyao
Turko, Illarion V
Mass spectrometry quantification of clusterin in the human brain
title Mass spectrometry quantification of clusterin in the human brain
title_full Mass spectrometry quantification of clusterin in the human brain
title_fullStr Mass spectrometry quantification of clusterin in the human brain
title_full_unstemmed Mass spectrometry quantification of clusterin in the human brain
title_short Mass spectrometry quantification of clusterin in the human brain
title_sort mass spectrometry quantification of clusterin in the human brain
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3470951/
https://www.ncbi.nlm.nih.gov/pubmed/22906254
http://dx.doi.org/10.1186/1750-1326-7-41
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