Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.

Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithio...

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Autores principales:	Luca Musante, Mayank Saraswat, Elodie Duriez, Barry Byrne, Alessandra Ravidà, Bruno Domon, Harry Holthofer
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ce954a7cfca647e1a95d8afd07f698f6
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spelling	oai:doaj.org-article:ce954a7cfca647e1a95d8afd07f698f62021-11-18T07:12:43ZBiochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.1932-620310.1371/journal.pone.0037279https://doaj.org/article/ce954a7cfca647e1a95d8afd07f698f62012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22808001/?tool=EBIhttps://doaj.org/toc/1932-6203Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.Luca MusanteMayank SaraswatElodie DuriezBarry ByrneAlessandra RavidàBruno DomonHarry HolthoferPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 7, p e37279 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Luca Musante Mayank Saraswat Elodie Duriez Barry Byrne Alessandra Ravidà Bruno Domon Harry Holthofer Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
description	Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.
format	article
author	Luca Musante Mayank Saraswat Elodie Duriez Barry Byrne Alessandra Ravidà Bruno Domon Harry Holthofer
author_facet	Luca Musante Mayank Saraswat Elodie Duriez Barry Byrne Alessandra Ravidà Bruno Domon Harry Holthofer
author_sort	Luca Musante
title	Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
title_short	Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
title_full	Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
title_fullStr	Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
title_full_unstemmed	Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.
title_sort	biochemical and physical characterisation of urinary nanovesicles following chaps treatment.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/ce954a7cfca647e1a95d8afd07f698f6
work_keys_str_mv	AT lucamusante biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment AT mayanksaraswat biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment AT elodieduriez biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment AT barrybyrne biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment AT alessandraravida biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment AT brunodomon biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment AT harryholthofer biochemicalandphysicalcharacterisationofurinarynanovesiclesfollowingchapstreatment
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Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment.

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