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Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS

Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc b...

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Autores principales: Dionisio, Giuseppe, Uddin, Mohammad Nasir, Vincze, Eva
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874762/
https://www.ncbi.nlm.nih.gov/pubmed/29342075
http://dx.doi.org/10.3390/proteomes6010003
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author Dionisio, Giuseppe
Uddin, Mohammad Nasir
Vincze, Eva
author_facet Dionisio, Giuseppe
Uddin, Mohammad Nasir
Vincze, Eva
author_sort Dionisio, Giuseppe
collection PubMed
description Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc binding proteins in different grain tissues, we microdissected barley grains into (1) seed coats; (2) aleurone/subaleurone; (3) embryo; and (4) endosperm. Initial screening for putative zinc binding proteins from the different tissue types was performed by fractionating proteins according to solubility (Osborne fractionation), and resolving those via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by polyvinylidene fluoride (PVDF) membrane blotting and dithizone staining. Selected protein fractions were subjected to Zn(2+)-immobilized metal ion affinity chromatography, and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have been captured by Zn-IMAC, and their zinc binding properties in relationship to the possibility of zinc storage is discussed.
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spelling pubmed-58747622018-04-02 Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS Dionisio, Giuseppe Uddin, Mohammad Nasir Vincze, Eva Proteomes Article Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc binding proteins in different grain tissues, we microdissected barley grains into (1) seed coats; (2) aleurone/subaleurone; (3) embryo; and (4) endosperm. Initial screening for putative zinc binding proteins from the different tissue types was performed by fractionating proteins according to solubility (Osborne fractionation), and resolving those via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by polyvinylidene fluoride (PVDF) membrane blotting and dithizone staining. Selected protein fractions were subjected to Zn(2+)-immobilized metal ion affinity chromatography, and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have been captured by Zn-IMAC, and their zinc binding properties in relationship to the possibility of zinc storage is discussed. MDPI 2018-01-17 /pmc/articles/PMC5874762/ /pubmed/29342075 http://dx.doi.org/10.3390/proteomes6010003 Text en © 2018 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
Dionisio, Giuseppe
Uddin, Mohammad Nasir
Vincze, Eva
Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS
title Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS
title_full Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS
title_fullStr Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS
title_full_unstemmed Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS
title_short Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS
title_sort enrichment and identification of the most abundant zinc binding proteins in developing barley grains by zinc-imac capture and nano lc-ms/ms
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874762/
https://www.ncbi.nlm.nih.gov/pubmed/29342075
http://dx.doi.org/10.3390/proteomes6010003
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