Ana Valéria Colnaghi Simionato

Determination of amino acids by capillary electrophoresis‐electrospray ionization‐mass spectrometry: An evaluation of different protein hydrolysis procedures

In this work, a CE equipment, online hyphenated to an IT MS analyzer by a linear sheath liquid interface promoting ESI, was used to develop a method for quantitative determination of amino acids. Under appropriate conditions (BGE composition, 0.8% HCOOH, 20% CH3OH; sheath liquid composition, 0.8% HCOOH, 60% methanol; VESI, +4.50 kV), analytical curves of all amino acids from 3 to 80 mg/L were recorded presenting acceptable linearity (r >0.99). LODs in the range of 16–172 μmol/L were obtained. BSA, a model protein, was submitted to different hydrolysis procedures (classical acid and basic, and catalyzed by the H+ form of a cation exchanger resin) and its amino acid profiles determined. In general, the resin‐mediated hydrolysis yields were overall similar or better than those obtained by classical acid or basic hydrolysis. The resulting experimental‐to‐theoretical BSA concentration ratios served as correction factors for the quantitation of amino acids in Brazil nut resin generated hydrolysates.

Detecção de sideróforos nas bactérias endofíticas Methylobacterium spp. associadas com Xylella fastidiosa subsp. pauca

The objective of this work was to study the production of siderophores by endophytic bacteria Methylobacterium spp., which occupy the same ecological niche as Xylella fastidiosa subsp. pauca (Xfp) in citrus plants. The siderophore production of Methylobacterium strains was tested according to chromeazurol agar assay test (CAS), Csaky test (hydroxamate-type) and Arnow test (catechol-type). In addition, the ability of Xfp to use siderophores, in vitro, produced by endophytic bacteria as source of iron, was evaluated. All 37 strains of Methylobacterium spp. tested were CAS-positive for siderophore production. Methylobacterium spp. produced hydroxamate-type, but not catechol-type siderophores. In vitro growth of Xfp was stimulated by the presence of supernatant siderophores of endophytic Methylobacterium mesophilicum.

CE‐MS and related techniques as a valuable tool in tumor biomarkers research

Cancer has been a disease of great concern because it is the second main cause of death in the world. Cures for most cancer pathologies have not yet been found, and an accurate and early diagnosis is essential for successful treatment. Therefore, research on tumor biomarkers has noticeably increased in recent years. The determination of such biomolecules, together with the routinely used laboratory exams for cancer diagnosis, would constitute a more reliable approach, known as systems biology. The “omics” era has corroborated in such investigations since the development of new technologies has arisen along with it. One of the techniques applied to the investigation of tumor biomarkers is CE, and the increasing applications of CE‐MS in this field are also observed. This review covers the published literature on tumor biomarker investigations by CE‐MS and related techniques, mostly within the last decade, but not limited to it. For didactic reasons this review is divided into the tumor biomarkers chemical classes, namely, proteins and related molecules, DNA adducts and modified nucleosides.

Eletroforese capilar acoplada à espectrometria de massas (CE-MS): vinte anos de desenvolvimento

CE-MS has been increasingly used for analysis of a vast array of compounds. This article reviews the different electrophoretic modes, interfaces and mass analyzers that are commonly used in the CE-MS coupling, as well as the technique advantages and performance characteristics. A large compilation of CE-MS applications is also presented. Therefore, this review is both a guide for beginners and a collection of key references for people who are familiar to the technique. Furthermore, this is the first CE-MS review published in a Brazilian journal and marks the installation of the first two commercial CE-MS units in Sao Paulo State.

Separation of Water‐Soluble Vitamins by Micellar Electrokinetic Capillary Chromatography in Pharmaceutical Samples

Abstract The separation of six water‐soluble vitamins (e.g., B1—thiamine, B2—riboflavin, B3—nicotinic acid, B6—pyridoxine, B12—cyanocobalamin, and C—ascorbic acid) was investigated using capillary electrophoresis. Initially, the most important variables in micellar electrokinetic capillary chromatography were established, such as: background electrolyte pH; surfactant concentration; detection wavelength; applied voltage, and amount of organic modifier. The solutes were separated within 8 min, with excellent efficiency (3 · 105 theoretical plates) and resolution, which means great advantage towards the analysis of real samples. Two pharmaceutical samples were analyzed using the optimized method, which was shown to be adequate for this purpose.

Unveiling differences between patients with acute coronary syndrome with and without ST elevation through fingerprinting with CE-MS and HILIC-MS targeted analysis.

Differences in the degree and severity of Acute Coronary Syndrome, associated to differences in the electrocardiogram, together with blood tests of biomarkers classify patients for diagnosis and treatment. Cases where the electrocardiogram and/or biomarkers are not conclusive still appear, and there is a need for complementary biomarkers for routine determinations. Metabolomics approaches with blind fingerprinting could reveal differences in metabolites, which must be confirmed by means of targeted determinations. CE‐MS and HILIC‐MS are well suited for the determination of highly polar compounds, like those from to the intermediate metabolism, altered due to acute stress induced by myocardial infarction. Serum from patients with ST‐elevated and non‐ST elevated myocardial infarction was collected at intensive care and emergency units, and fingerprinted with CE‐MS. Data pretreatment and analysis showed up carnitine‐related compounds and amino acids differentially present in both groups. Acylcarnitines and amino acids were then quantitatively measured with HILIC‐MS‐QqQ. The significance of the differences and the sensitivity/specificity of each compound were individually evaluated. The ratio of free carnitine to acylcarnitines, together with the ratios of acetylcarnitine to betaine, to threonine, and to citrulline, showed high significance and area under the curve in the respective receiver operating characteristic curves. This study opens new possibilities for defining new sets of biomarkers for refining the diagnosis of the patients with difficult classification.

Characterization of protein hydrolysates of cosmetic use by CE‐MS

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid--pH 1.8--in 70:30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75:25-25:75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.

Evidences of siderophores synthesis by Grapevine Xylella fastidiosa, causal agent of pierce's disease, through instrumental approaches

Siderophore molecules from grapevine Xylella fastidiosa were investigated. Such metabolites sequester iron, an essential element, from the host, making them a potential factor of pathogenicty. In an iron-limited medium, siderophores were detected in culture plates of X. fastidiosa containing the complex Chromeazurol S (CAS). A combination of different instrumental analyses was used for siderophore(s) characterization, such as: immobilized metal affinity chromatography (IMAC), micelar electrokinetic capillary chromatography (MEKC) and electrospray-mass spectrometry (ESI-MS). The results show that grapevine X. fastidiosa produced siderophore(s), as confirmed by the CAS plate assay. The extraction of the compound(s) using IMAC with immobilized Fe3+ was important for analyte purification. The chelator was not separated by capillary zone electrophoresis, indicating the possibility of a neutral compound under the investigated pHs. MEKC runs presented a different peak (when compared to the control analysis) which represented a slightly hydrophobic compound. Mass spectrometry showed that the compound(s) may have a relative molecular mass within the expected range for siderophore molecules, such as 875, 1004 and 1092 Da.

Assessment Of Nucleosides As Putative Tumor Biomarkers In Prostate Cancer Screening By Ce-uv

AbstractCancer is responsible for millions of deaths worldwide, but most base diseases may be cured if detected early. Screening tests may be used to identify early-stage malignant neoplasms. However, the major screening tool for prostate cancer, the prostate-specific antigen test, has unsuitable sensitivity. Since cancer cells may affect the pattern of consumption and excretion of nucleosides, such biomolecules are putative biomarkers that can be used for diagnosis and treatment evaluation. Using a previously validated method for the analysis of nucleosides in blood serum by capillary electrophoresis with UV–vis spectroscopy detection, we investigated 60 samples from healthy individuals and 42 samples from prostate cancer patients. The concentrations of nucleosides in both groups were compared and a multivariate partial least squares–discriminant analysis classification model was optimized for prediction of prostate cancer. The validation of the model with an independent sample set resulted in the correct classification of 82.4% of the samples, with sensitivity of 90.5% and specificity of 76.7%. A significant downregulation of 5-methyluridine and inosine was observed, which can be indicative of the carcinogenic process. Therefore, such analytes are potential candidates for prostate cancer screening. Graphical AbstractSeparation of the studied nucleosides and the internal standard 8-Bromoguanosine by CE-UV (a); classification of the external validation samples (30 from healthy volunteers and 21 from prostate cancer patients) by the developed Partial Least Square – Discriminant Analysis (PLS-DA) model with accuracy of 82.4% (b); Receiver Operating Characteristics (ROC) curve (c); and Variable Importance in the Projection (VIP) values for the studied nucleosides (d). A significant down-regulation of 5- methyluridine (5mU) and inosine (I) was observed, which can be indicative of the presence of prostate tumors.

Development and validation of a micellar electrokinetic capillary chromatographic method for the assessment of nucleosides, potential biomarkers, in blood serum

Malignant tumors are the cause of millions of deaths all over the world every year, but the detection of the disease in the first stages may save lives. Tumor biomarkers allow earlier and less invasive diagnosis, hence they are vital in the cancer treatment. Nucleosides have been investigated as a potential group of tumor biomarkers present in biological fluids. In this work, a method for the analysis of nucleosides in blood serum samples by capillary electrophoresis with UV detection (CE-UV) was developed and validated according to the current legislation in Brazil. Separation of ten nucleosides plus the internal standard was achieved in ca. 25 min. The method may contribute for earlier and more accurate diagnosis of cancer cases.