Gustavo de Souza Pessôa

Comparative proteomics and metallomics studies in Arabidopsis thaliana leaf tissues: Evaluation of the selenium addition in transgenic and nontransgenic plants using two-dimensional difference gel electrophoresis and laser ablation imaging

The main goal of this work is to evaluate some differential protein species in transgenic (T) and nontransgenic (NT) Arabidopsis thaliana plants after their cultivation in the presence or absence of sodium selenite. The transgenic line was obtained through insertion of CaMV 35S controlling nptII gene. Comparative proteomics through 2D‐DIGE is carried out in four different groups (NT × T; NT × Se‐NT (where Se is selenium); Se‐NT × Se‐T, and T × Se‐T). Although no differential proteins are achieved in the T × Se‐T group, for the others, 68 differential proteins (by applying a regulation factor ≥1.5) are achieved, and 27 of them accurately characterized by ESI‐MS/MS. These proteins are classified into metabolism, energy, signal transduction, disease/defense categories, and some of them are involved in the glycolysis pathway—Photosystems I and II and ROS combat. Additionally, laser ablation imaging is used for evaluating the Se and sulfur distribution in leaves of different groups, corroborating some results obtained and related to proteins involved in the glycolysis pathway. From these results, it is possible to conclude that the genetic modification also confers to the plant resistance to oxidative stress.

Laser ablation and inductively coupled plasma mass spectrometry focusing on bioimaging from elemental distribution using MatLab software: a practical guide

The parameters influencing laser ablation inductively coupled plasma mass spectrometry as a tool for imaging elemental distribution in tissues are critically commented on in this work, and penile cancer tissue was used as a model. General aspects of LA-ICP-MS are discussed, and among them, issues regarding sample preparation and calibration. The optimization process of the following variables is described in detail in a step-by-step tutorial manner: laser intensity, laser frequency, laser resolution, ICP radiofrequency, nebulizer and auxiliary gas flow rates, and C isotopes as internal standards. Once the best condition is found for each variable, MATLAB software is used to generate two-dimensional images. Herein it is also explained how to use MATLAB software to generate tissue images using acquisition, exporting and data treatment parameters.

LA-iMageS: a software for elemental distribution bioimaging using LA–ICP–MS data

The spatial distribution of chemical elements in different types of samples is an important field in several research areas such as biology, paleontology or biomedicine, among others. Elemental distribution imaging by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) is an effective technique for qualitative and quantitative imaging due to its high spatial resolution and sensitivity. By applying this technique, vast amounts of raw data are generated to obtain high-quality images, essentially making the use of specific LA–ICP–MS imaging software that can process such data absolutely mandatory. Since existing solutions are usually commercial or hard-to-use for average users, this work introduces LA-iMageS, an open-source, free-to-use multiplatform application for fast and automatic generation of high-quality elemental distribution bioimages from LA–ICP–MS data in the PerkinElmer Elan XL format, whose results can be directly exported to external applications for further analysis. A key strength of LA-iMageS is its substantial added value for users, with particular regard to the customization of the elemental distribution bioimages, which allows, among other features, the ability to change color maps, increase image resolution or toggle between 2D and 3D visualizations.

Ion mobility mass spectrometry: an elegant alternative focusing on speciation studies

This work is proposed to demonstrate the Traveling-Wave Ion Mobility Specrometry (TWIMS) coupled to Mass Spectrometry (MS) as an alternative technique for speciation analysis between metals/metalloids and biomolecules. Mobilities of bovine carbonic anhydrase bound to Ba2+, Cu2+, Pb2+, Zn2+, Cr3+, Cr6+, Se4+ and Se6+ were estimated. The metal belonging to the bovine carbonic anhydrase structure, commonly found in the commercially available enzyme, was removed by filtration, using centrifugal filter devices. Then, some metals/metalloids were added to 10.0 mmol L−1 ammonium acetate at pH = 6.8 enzyme solution. Experiments were carried out by direct insertion of the sample at 10 μL min−1 flow rate into the ESI source of the instrument. Carbonic anhydrase mobility varied according to the metal bound in its structure, following the order: Zn2+ < Cu2+ < Ba2+ < Pb2+. Metals with higher affinity by the enzyme, such as Zn2+ and Cu2+ had lower mobility, suggesting a higher structural modification, binding itself to the enzyme metallic site. Considering metals with different oxidation states, the enzyme mobility followed the order: Se4+ < Cr6+ < Se6+ < Cr3+.

The importance of evaluating the real metal concentration in nanoparticles post-synthesis for their applications: A case-study using silver nanoparticles.

To determine whether the effect observed in a study is related to the nanoparticle only or to their synergic effect with the free metal ions, the real concentration of silver (104±8 and 100±2 mg L(-1)) after AgNP synthesis is obtained through ICP-MS and ICP OES in the solution after the AgNP synthesis and in different fractions after centrifugation (at 8100 g for 40 min). From the resuspension of the AgNP contained in the solution (AgNP-total) after synthesis (AgNP-res), concentrations of 49±3 and 51±3 mg L(-1) are found and concentrations of 50±7 and 47±2 mg L(-1) in the supernatant (Ag-sup) are found using ICP-MS and ICP OES respectively. The characterization of AgNP-total, AgNP-res and Ag-sup is performed by HRTEM and UV-vis, corroborating the results in terms of Ag determination, and indicates that half of the total silver concentration is in the AgNP form and that the other half is in the free silver form. The results of the stability test of the NPs indicate a 7% decrease in Ag as NP three months after its synthesis.

A quantitative approach for Cd, Cu, Fe and Mn through laser ablation imaging for evaluating the translocation and accumulation of metals in sunflower seeds

The uptake and accumulation of Cd in sunflower seeds represents an important pathway for imputing potentially toxic metals into human and animal food. In this way, bioimaging of Cd and micronutrients (Cu, Fe and Mn) in the seeds of sunflower grown in soil contaminated with Cd are performed. For this task, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is used in quantitative approach, considering four groups: precursor, control, Cd-low (50mg) and Cd-high (700mg). For attaining our proposals, ICP-MS (nebulizer and auxiliary flow rates and radiofrequency power) and LA (laser intensity, frequency and spot size) parameters were optimized, and the analytical signal improved to 197%, 217%, 232%, and 283%, for 57Fe, 112Cd, 55Mn and 63Cu, respectively. The accuracy of proposed method using LA-ICP-MS is evaluated comparing the CRM results (Tomato leaves, NIST SRM 1573a). No difference is found at 95% confidence level. Regarding Cd accumulation in sunflower seeds, the results indicated that cadmium is translocated to seeds, and the cotyledons showed the highest concentration (Cd-high group), ranging from 10 to 20µgg-1. Considering both total concentration and the distribution in the seeds, Cd uptake is responsible to the homeostasis misbalance of micronutrients, which play an essential role in the sunflower metabolism. Such results highlight the importance of bioimaging evaluation, in the translocation and accumulation of metals, contributing to expand the information available of this culture.

Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques

The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL-1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL-1. Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers.

Proteomics strategies for bipolar disorder evaluation: From sample preparation to validation.

Bipolar disorder (BD) is a complex and costly psychiatric disorder, which affects one hundred million people worldwide. Due to its heterogeneity, correct BD diagnosis is still a challenge. In order to overcome this issue, different bioanalytical strategies have been proposed in the literature recently. Among these strategies, proteomic approaches have arisen as some of the most promising in the area. Thus, recent applications suggest protein profiles to further refine the proteome of BD as well as the discovery of novel protein biomarkers to facilitate diagnostics. In this review, the state-of-art of proteomic research in BD is summarized. Furthermore, important aspects of proteomics for understanding of BD, such as sample type and size, sampling, sample preparation, gel-based and gel-free proteomics, proteomic quantitative and protein validation are overviewed.

Incorporation of 67Zn and 68Zn into carbonic anhydrase: effects on isotope enrichment and enzymatic aspects

An enrichment of carbonic anhydrase (CA) with zinc isotopes (67Zn and 68Zn) is carried out to evaluate protein binding with a specific isotope. For this task, apo-CA is prepared with ortho-phenanthroline (OP) thus formatting the Zn–OP complex, and allowing the study of metal binding at CA. After an ultrafiltration procedure, the Zn–OP complex is removed, and the apo-CA is obtained, which is used for incubation with different zinc isotopes. An inductively coupled plasma mass spectrometer equipped with a dynamic reaction cell (DRC) is used for speciation purposes focusing on isotope ratio measurements. The optimization of instrumental parameters results in trueness measurements closer to natural conditions for Zn isotope ratios. The obtained precision is within the range observed for the same kind of m/z analyzer. To address the purity of apo-carbonic anhydrase, the protein was chromatographically separated using size exclusion with UV/Vis and ICP-MS detection. The incubation of CA with Zn isotopes changes the natural abundance of zinc isotopes, increasing the isotope ratios by 3.0 and 1.4 times for 67Zn and 68Zn, respectively, when compared to IRMM-3702. Such Zn-isotopically enriched CA was characterized, containing 1 atom of Zn per mole of CA. In terms of enzymatic activity, CA is enriched with different Zn isotopes yielding similar statistical results, producing 283 ± 32 nmol, 283 ± 11 nmol and 259 ± 32 nmol of p-nitrophenol for CA incubated with IRMM-3702, 67Zn and 68Zn, respectively. As significant differences were observed among apo and holo forms of CA, the enriched protein showed great potential for use in speciation analysis studies.

Elaboração de estratégias analíticas para a síntese da droga “chá de pilha” e a determinação de metais utilizando a técnica de ICP OES e ICP MS

Resumo: Este trabalho teve como foco a determinação da quantidade de Mn, Co, Li, Ni, Zn, Hg e Pb presentes na droga de abuso caseira chá de pilha, onde uma infusão de baterias é feita, filtrada e depois ingerida. Esta droga vem sendo extensivamente usada por usuários de drogas de baixa renda em momentos de abstinência. A análise dos metais foi feita utilizando a técnica de ICP OES com padrão interno de Rh e posteriormente confirmada por análise em um equipamento ICP MS. Analisou-se a extração dos metais em 10 e 30 min. de infusão. Houve um cuidado na elaboração dos métodos de extração para que não se usasse reagentes de alta toxicidade já que o chá, depois de elaborado, seria consumido pelo usuário da droga em uma situação hipotética. Também se deu atenção para que fossem usados na equipamentos e reagentes acessíveis, já que esta é uma droga caseira. Obteve-se concentrações maiores que a máxima permitida pelo padrão de potabilidade da água exigido pela CETESB para Mn, Zn e Ni.