Enrichment and identification of the most abundant zinc binding proteins in developing barley grains by Zinc-IMAC capture and nano LC-MS/MS

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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. / Dionisio, Giuseppe; Uddin, Mohammad Nasir; Vincze, Eva.

I: Proteomes, Bind 6, Nr. 1, 3, 17.01.2018.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{7290223d6ed649898767f482e4d7485a,
title = "Enrichment and identification of the most abundant zinc binding proteins in developing barley grains by Zinc-IMAC capture and nano LC-MS/MS",
abstract = "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 Zn2+-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.",
keywords = "metal-binding proteins, Zn-IMAC, mass spectrometry",
author = "Giuseppe Dionisio and Uddin, {Mohammad Nasir} and Eva Vincze",
year = "2018",
month = jan,
day = "17",
doi = "10.3390/proteomes6010003",
language = "English",
volume = "6",
journal = "Proteomes",
issn = "2227-7382",
publisher = "MDPI AG, Basel, Switzerland",
number = "1",

}

RIS

TY - JOUR

T1 - Enrichment and identification of the most abundant zinc binding proteins in developing barley grains by Zinc-IMAC capture and nano LC-MS/MS

AU - Dionisio, Giuseppe

AU - Uddin, Mohammad Nasir

AU - Vincze, Eva

PY - 2018/1/17

Y1 - 2018/1/17

N2 - 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 Zn2+-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.

AB - 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 Zn2+-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.

KW - metal-binding proteins

KW - Zn-IMAC

KW - mass spectrometry

U2 - 10.3390/proteomes6010003

DO - 10.3390/proteomes6010003

M3 - Journal article

C2 - 29342075

VL - 6

JO - Proteomes

JF - Proteomes

SN - 2227-7382

IS - 1

M1 - 3

ER -