Metalômica comparativa de soja [Glycine max (L.) [Glycine max (L.) merrill] using a multidimensional separation system

Author(s)

Lidiane Raquel Verola Mataveli

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2013-05-24

Examining board members

Marco Aurelio Zezzi Arruda; Fernando Barbosa Júnior; Pedro de Magalhães Padilha; Ana Valéria Colnaghi Simionato; Lauro Tatsuo Kubota

• Advisor: Ljubica Tasic; Marco Aurelio Zezzi Arruda

Abstract

This work consisted of comparative metallomics studies of transgenic (T) and nontransgenic (NT) soybean seeds. To that end, T and NT seeds were compared at first taking into account: the total concentration of the elements, the elements behavior during extraction using sequential fractionation, and bioacessibility of the elements after simulated gastrointestinal digestion procedure (in vitro). Preliminary analyzes were done using ICP-MS with quadrupole mass analyzer, and the subsequent analysis using high-resolution inductively coupled plasma mass spectrometry, (HR-ICP-MS). 25 elements were determined at concentrations ranging from 1 ng g up to %. It was observed that T and NT soybean seeds exhibit statistically significant differences in the concentrations of Cu, Fe and Sr, the first two having higher concentrations in the T seeds, and the last with the highest concentration in soybeans NT. These results were also reflected in the contents of these elements in aqueous extracts and residues obtained through sequential fractionation. Also, the contributions of bioaccessible fractions of Cu, Fe and other elements (Mn, S, Zn) to the total content of the elements in T soybean seeds were higher than those found in NT soybean seeds. Subsequently, studies were continued using bidimensional liquid chromatography, the first dimension consisting of size exclusion chromatography (SEC) and the second dimension of anion exchange chromatography (AEX). In the first chromatographic dimension three fractions containing metals were identified using hyphenation SEC-ICP-MS, the first corresponding to molar masses between 38.1 and 181.1 kDa, the second corresponding to molar masses between 8.2 and 17.2 kDa and the third fraction corresponding to molar masses between 0.4 and 3.8 kDa. The three identified fractions were separated and lyophilized, and again separated using anion exchange chromatography (AEX). Metals were found in all the separated fractions by SEC: three sub-fractions of the first fraction, a subfraction in the second fraction and three sub-fractions in the third fraction. These peaks were collected, lyophilized, digested and taken to the mass spectrometer for protein identification. 33 proteins were identified, and, among them, two proteins previously related to metals were found: seed lipoxygenase 1 and b-conglycinin. After chromatographic separation in the second dimension, the resultant subfractions were lyophilized and subjected to a third separation dimension using onedimensional polyacrylamide gel electrophoresis (SDS-PAGE). After the separation, the bands were cut out and digested to examine the metals contained therein, highlighting the results obtained for Fe, which was quantified in the bands of the sub-fraction where the peak of the element is found in the analysis by ICP-AEX - MS. Also, the bands were digested triptically to identify the proteins, and once again proteins associated to metals were identified: 3-lipoxygenase A chain (Soybean) complex with 13 (S)-Hydroperoxy-9 (Z), 11 ( E)-octadecadienoic acid, beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max] seed lipoxygenase-2 (Pisum sativum) and beta-conglycinin (AU)

• FAPESP's process: 08/03981-4 - Comparative metallomics of transgenic and non-transgenic soybeans (Glicine max L. Merril)using bidimensional separation systems
• Grantee: Lidiane Raquel Verola Mataveli
• Support Opportunities: Scholarships in Brazil - Doctorate