Sensitive determination of metalloprotein in salt-rich matrices by size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry.

Metalloproteins play crucial and distinct roles in a variety of biological processes that rely heavily on the metal ions and various proteins. However, there is still a lack of method for rapid analysis of metalloproteins in complex samples, especially in salt-rich matrices. In this study, a sensitive method for separation and determination of metalloproteins in salt-rich matrices was developed based on the size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry (SEC-ICP-MS), combining with the high matrix introduction (HMI) mode, which is quite essential for biological system. The separation conditions of the SEC-ICP-MS system were optimized by using four iodine labeled proteins with different molecular weights, including bovine serum albumin (BSA, 66.0 kDa), ovalbumin (OVA, 44.0 kDa), carbonic anhydrase (CA, 29.0 kDa) and ribonuclease A (RA, 13.7 kDa). After optimization, four iodine labeled proteins and iodine ions were successfully separated within 30 min by using 10 mmol/L HEPES and 40 mmol/L Na2SO4 (pH=7.0) as mobile phase and a linear relationship between log molecular weight and retention time was established. The relative standard deviations (RSDs, n = 5) of the retention time and peak areas for the four iodine labeled proteins were in the range of 0.2-0.9% and 3.3-7.7%, respectively, suggesting good precision and repeatability. Then the proposed method was successfully applied to the rapid separation and detection of lead-binding proteins in real biological tissue samples.

Metalloproteomics for Biomedical Research: Methodology and Applications.

Metals are essential components in life processes and participate in many important biological processes. Dysregulation of metal homeostasis is correlated with many diseases. Metals are also frequently incorporated into diagnosis and therapeutics. Understanding of metal homeostasis under (patho)physiological conditions and the molecular mechanisms of action of metallodrugs in biological systems has positive impacts on human health. As an emerging interdisciplinary area of research, metalloproteomics involves investigating metal-protein interactions in biological systems at a proteome-wide scale, has received growing attention, and has been implemented into metal-related research. In this review, we summarize the recent advances in metalloproteomics methodologies and applications. We also highlight emerging single-cell metalloproteomics, including time-resolved inductively coupled plasma mass spectrometry, mass cytometry, and secondary ion mass spectrometry. Finally, we discuss future perspectives in metalloproteomics, aiming to attract more original research to develop more advanced methodologies, which could be utilized rapidly by biochemists or biologists to expand our knowledge of how metal functions in biology and medicine.

Unravelling neurological disorders through metallomics-based approaches.

Understanding the biological process involving metals and biomolecules in the brain is essential for establishing the origin of neurological disorders, such as neurodegenerative and psychiatric diseases. From this perspective, this critical review presents recent advances in this topic, showing possible mechanisms involving the disruption of metal homeostasis and the pathogenesis of neurological disorders. We also discuss the main challenges observed in metallomics studies associated with neurological disorders, including those related to sample preparation and analyte quantification.

Effects of white, yellow, and blue colored LEDs on milk production, milk composition, and physiological responses in dairy cattle.

Light emitting diode (LED) is more energy efficient than incandescent or fluorescent light. This study was to evaluate effects of different colored LEDs on milk production, milk composition, and physiology of Holstein cow. According to milk production and parity, cows (n = 186) were allotted to four treatments: control (natural daylight), white, yellow, and blue LED groups. Of these, 40 cows that had passed 57 day-in-milk were used. Yellow and blue LED groups demonstrated greater rates of decline in milk production than control and white LED groups. At the finish point, milk fat, protein, and lactose contents were the lowest in the blue LED group, whereas milk-urea-nitrogen levels were the highest in the yellow and blue LED groups. Extended exposure to blue LED light lowered antioxidant enzyme activity and insulin-like growth factor-1 levels. Prolactin concentrations were higher in the white and blue LED groups than in the control. Cortisol level was the highest in the blue LED group among the groups. Nonesterified fatty acid levels in the yellow and blue LED groups decreased to the greatest extent compared to the start point. These results suggest that blue LED light can decrease milk production and generate more stress than white and yellow LED lights.

Choice of mobile phase: Implications for size exclusion chromatography-inductively coupled plasma-mass spectrometry analyses of copper, zinc and iron metalloproteins.

The correct identification of the metalloproteins present in human tissues and fluids is essential to our understanding of the cellular mechanisms underpinning a host of health disorders. Separation and analysis of biological samples are typically done via size exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry (SEC-ICP-MS). Although this technique can be extremely effective in identification of potential metalloproteins, the choice of mobile phase may have a marked effect on results, results by adversely affecting metal-protein bonds of the metalloproteins of interest. To assess the choice of mobile phase on SEC-ICP-MS resolution and the resulting metalloproteome pattern, we analysed several different sample types (brain homogenate; Cu/Zn-superoxide dismutase (SOD1); a molecular weight standard mix containing ferritin (Ft), ceruloplasmin (Cp), cytochrome c (CytC), vitamin B12 (B12) and thyroglobulin (Tg) using six different mobile phase conditions (200 mM, pH 7.5 solutions of ammonium salts nitrate, acetate, and sulfate; HEPES, MOPS and Tris-HCl). Our findings suggest that ammonium nitrate, ammonium acetate and Tris-HCl are optimal choices for the mobile phase, with the specific choice being dependent on both the number of samples and method of detection that is hyphenated with separation. Furthermore, we found that MOPS, HEPES and ammonium sulfate mobile phases all caused significant changes to peak resolution, retention time and overall profile shape. MOPS and HEPES, in particular, produced additional Fe peaks that were not detected with any of the other mobile phases that were investigated. As well as this, MOPS and HEPES both caused significant concentration dependent matrix suppression of the internal standard.

Prognostic significance of TRIM59 for cancer patient survival: A systematic review and meta-analysis.

BACKGROUND: The family of tripartite motif (TRIM) proteins, which includes 80 known TRIM protein genes in humans, play a key role in cellular processes. TRIM59, a member of the TRIM family of proteins, has been reported to be involved in the carcinogenesis of multiple types of tumors. However, the prognostic value of TRIM59 in the survival of tumor patients remains controversial. We therefore conducted a meta-analysis to assess the prognostic significance of TRIM59 in cancer patients. MATERIALS AND METHODS: PubMed, Embase, VIP, CNKI and Wanfang Data were searched for eligible reports published before September 30, 2018. The hazard ratio (HR) and 95% confidence intervals (CIs) were adopted to estimate the association between TRIM59 and overall survival (OS). RESULTS: Six studies with 1584 patients were included to assess the effect. The results showed that high levels of TRIM59 were significantly associated with poor OS in cancer patients (HR = 1.43, 95%CI: 1.24-1.66, P < .001), indicating that higher TRIM59 expression could be an independent prognostic factor for poor survival in cancer patients. CONCLUSION: Our meta-analysis suggests that higher TRIM59 expression predicts poor prognosis in cancer patients, and it may therefore serve as a promising prognostic factor.

Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye.

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol.

Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.

3D printed gel electrophoresis device coupling with ICP-MS for online separation and detection of metalloproteins.

We successfully developed a strategy to combine a customized gel electrophoresis device with ICP-MS for online separation and detection of metalloproteins. The self-designed horizontal column gel electrophoresis device was rapidly and easily fabricated in the laboratory via 3D printing with a low cost. The feasibility of 3D printing to fabricate this device was investigated by offline separation of commercial protein standards. And a better separation efficiency was found when using gel tubes printed with higher printing precision. As a proof-of-concept, the performance of the whole system is demonstrated by online separation and detection of both iodinated protein standards and proteins in rat blood plasma samples. Benefits from 3D printing, customized modification or further optimization can be readily achieved for a better protein separation and detection efficiency.

Identification of Biomarkers of Mercury Contamination in Brachyplatystoma filamentosum of the Madeira River, Brazil, Using Metalloproteomic Strategies.

Predator fish can accumulate high levels of mercury, which qualifies them as potential indicators of this toxic metal. The predatory species Brachyplatystoma filamentosum, popularly known as filhote, is among the most consumed species in the Brazilian Amazon. Continuing the metalloproteomic studies of mercury in Amazonian fishes that have been developed in the last 5 years, the present paper provides the data of protein characterization associated with mercury in muscle and liver samples of filhote (Brachyplatystoma filamentosum) collected in the Madeira River, Brazilian Amazon. The mercury concentration in the muscle and liver samples was determined by graphite furnace atomic absorption spectrometry (GFAAS). The protein fraction was extracted in an aqueous medium, and later, a fractional precipitation procedure was performed to obtain the protein pellets. Then, the proteome of the tissue samples of this fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and a mercury mapping of the protein spots was carried out by GFAAS after acid digestion. Protein spots that had mercury were characterized by mass spectrometry with electrospray ionization in sequence (ESI-MS/MS) after tryptic digestion. It was possible to characterize 11 mercury-associated protein spots that presented biomarker characteristics and could be used to monitor mercury in fish species of the Amazon region. Thus, the metalloproteomic strategies used in the present study allowed us to characterize 11 mercury-associated protein spots. It should be noted that the protein spots identified as GFRP, TMEM186, TMEM57B, and BHMT, which have coordination sites for elements with characteristics of soft acids, such as mercury, can be used as biomarkers of mercury contamination in monitoring studies of this toxic metal in fish species from the Amazon region.

Across the spectrum: integrating multidimensional metal analytics for in situ metallomic imaging.

To know how much of a metal species is in a particular location within a biological context at any given time is essential for understanding the intricate roles of metals in biology and is the fundamental question upon which the field of metallomics was born. Simply put, seeing is powerful. With the combination of spectroscopy and microscopy, we can now see metals within complex biological matrices complemented by information about associated molecules and their structures. With the addition of mass spectrometry and particle beam based techniques, the field of view grows to cover greater sensitivities and spatial resolutions, addressing structural, functional and quantitative metallomic questions from the atomic level to whole body processes. In this perspective, I present a paradigm shift in the way we relate to and integrate current and developing metallomic analytics, highlighting both familiar and perhaps less well-known state of the art techniques for in situ metallomic imaging, specific biological applications, and their use in correlative studies. There is a genuine need to abandon scientific silos and, through the establishment of a metallomic scientific platform for further development of multidimensional analytics for in situ metallomic imaging, we have an incredible opportunity to enhance the field of metallomics and demonstrate how discovery research can be done more effectively.

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.

Two-dimensional (weak anion exchange chromatography-gel electrophoresis) separations coupling to inductively coupled plasma mass spectrometry strategy for analysis of metalloproteins.

A two-dimensional (2-D, weak anion exchange chromatography-gel electrophoresis, WAX-GE) separations coupling to inductively coupled plasma mass spectrometry (ICP-MS) strategy was developed for efficient tracking of metalloproteins. Samples were fractionized with the primary dimension (WAX) and the collected fractions were subsequently subjected to the secondary dimension (GE) and finally detected with ICP-MS. The metalloproteins of interest from the secondary dimension were online split and collected for further manipulation. The molecular weight of metalloprotein was calibrated with a home-made protein marker including six iodine-labelled proteins with molecular weights ranging from 14 kDa to 77 kDa. The developed 2-D system is of higher separation resolution as compared to one-dimensional (1-D) system. Metalloproteins in Escherichia coli were further examined for validation of the method preformation. Several known or unknown metal-associated proteins were identified, evidencing the feasibility of the proposed method. Taken together, we established a solid methodology for efficient separation and qualitative study of metalloproteomics, providing a new strategy for the metallomics research.

Metalloproteomics Approach to Analyze Mercury in Breast Milk and Hair Samples of Lactating Women in Communities of the Amazon Basin, Brazil.

Mercury is a potentially toxic element that is present in the environment of the Brazilian Amazon and is responsible for adverse health effects in humans. This study sought to assess possible protein biomarkers of mercury exposure in breast milk samples from lactating women in the Madeira and Negro Rivers in the Brazilian Amazon. The mercury content of hair samples of lactating women was determined, and the proteome of breast milk samples was obtained using two-dimensional electrophoresis after protein precipitation with acetone. Mercury measurements of protein spots obtained via protein fractionation were performed by graphite furnace atomic absorption spectrometry (GFAAS), and it was observed that mercury is linked to proteins with molecular masses in the range of 14-26 kDa. The total mercury concentration was also determined by GFAAS in unprocessed milk, lyophilized milk, and protein pellets, with the purpose of determining the mercury mass balance in relation to the concentration of this element in milk and pellets. Approximately 85 to 97% of mercury present in the lyophilized milk from samples of lactating women of the Madeira River is bound in the protein fraction. From lactating women of the Negro River, approximately 49% of the total mercury is bound in the protein fraction, and a difference of 51% is bound in the lipid fraction.

Identification of the zinc, copper and cadmium metalloproteome of the protozoon Tetrahymena thermophila by systematic bioinformatics.

Tetrahymena thermophila (T. thermophila) is a ciliated protozoon that can detect freshwater pollution by heavy metals ("whole-cell biosensor"). This work employed a systematic bioinformatics approach to predict and analyze the metalloproteome of T. thermophila for the essential Zn, Cu and the non-essential Cd. 3784 metal-binding proteins were identified compared to the 456 annotated so far in UniProt. The localization, functional classification, and the functionally enriched network of the newly identified metalloproteome are presented. Cd toxicity could be explained in terms of the metal replacing Cu and especially Zn in MAPKs, transporters and antioxidant enzymes. The predicted results for Cd toxicity and responses reflect those observed experimentally in different organisms after their exposure to Cd.

Quistes renales y aneurismas de aorta de gran tamaño / Renal cysts and large aortic aneurysms

INTRODUCCIÓN: Los quistes renales se han relacionado con una mayor presencia y un mayor diámetro de aneurismas de aorta abdominal (AAA). OBJETIVO: Evaluar la proporción de pacientes con AAA y con quiste simple renal (QSR) en nuestra población y valorar su relación con el diámetro aneurismático. MATERIAL Y MÉTODOS: Realizamos un estudio transversal de pacientes consecutivos diagnosticados de AAA infrarrenal con tomografía axial computarizada con contraste entre 2013 y 2016 en nuestro centro. Registramos sus datos demográficos, factores de riesgo para el desarrollo y el crecimiento del AAA y la presencia de QSR. Se realizó una estadística descriptiva con medidas de tendencia central, dispersión y un análisis de la relación del diámetro aneurismático con la presencia de QSR y su presencia uni- o bilateral. RESULTADOS: Incluimos a 135 pacientes con edad media de 74 años (DE: 11,3). El 54,8% (n=68) tenían QSR, de los cuales el 50,7% (n=35) eran bilaterales. El diámetro medio de los AAA de los pacientes sin QSR (n=67) fue de 59,2mm (DE: 12,3) en contraste con el diámetro aneurismático medio de los pacientes con QSR (n=68) que fue de 65,2mm (DE: 15,3) (p = 0,36). Tampoco se observaron diferencias significativas en el diámetro máximo de los AAA de los pacientes con QSR unilaterales (n=33) respecto a los pacientes con AAA y QSR bilaterales (n=35) (53,5 versus 59,1mm; p = 0,16). CONCLUSIÓN: En nuestra serie, no se encontró relación significativa entre presencia de QSR, bilateralidad y tamaño de los AAA

INTRODUCTION: Renal cysts have been associated with an increase in the presence abdominal aortic aneurysm (AAA) and also with a larger aneurysmal sac diameter. OBJECTIVE: To study the proportion of patients with AAA and simple renal cyst (SRC) in our population and study their relationship with the aneurysmal diameter. MATERIAL AND METHODS: A cross-sectional study was conducted on consecutive patients diagnosed with infrarenal AAA using contrast computed tomography between 2013 and 2016 in our centre. Information was collected on the demographics of participants, including risk factors associated with AAA development, growth rates of AAA, and the presence of renal cysts. Descriptive statistics were performed with measurements of central tendency and dispersion, and an analysis of the relationship between aneurysmal diameter and the presence of uni- or bilateral renal cysts. RESULTS: A total of 135 patients were included, with a mean age of 74 years (SD 11.3). Renal cysts were present in 54.8% (n=68) of cases, with 50.7% (n=35) of these being bilateral SRC. The mean diameter of the AAA in patients (n=67) without a SRC was 59.2mm (SD 12.3), as opposed to a mean AAA diameter of 65.2mm (SD 15.3) in patients (n=68) with a SRC (P=.36). No significant differences were observed in the maximum diameter of the AAA in patients (n=33) with a unilateral SRC compared to patients with a bilateral SRC (n=35) (53.5 versus 59.1mm; P=.16). CONCLUSION: No significant relationship was found in this series between the presence of an SRC, bilateralism, and the size of the AAA

In vivo identification of potential uranium protein targets in zebrafish ovaries after chronic waterborne exposure.

Ecotoxicological studies have indicated the reprotoxicity of uranium (U) in zebrafish, but its molecular mechanisms remain unclear. Due to the non-covalent nature of U-protein complexes, canonical proteomics approaches are often not relevant as they usually use denaturating reagents or solvents. In this study, non-denaturating (ND) methods were used to obtain insight into the nature of U potential targets in ovaries of reproduced and non-reproduced zebrafish after 20 days of exposure to an environmentally relevant U concentration (20 µg L-1). After the ND sample preparation, 1-dimensional (SEC) and 2-dimensional (OGE × SEC) separations followed by ICP-sector-field MS measurements (U, P, Fe, Cu, and Zn) enabled the determination of chemical characteristics (MW, pI) of the metal-protein complexes. Phosphorus and U coelution confirmed the affinity of U for P-containing proteins. In addition, 2D separation allowed the discrimination of Fe-metalloproteins as potential U targets. Finally, 20 protein candidates for U complexation were identified after tryptic digestion conditions by LC-ESI FT MS and a database search. Potential U targets were mainly involved in three biological processes: oxidative stress regulation (SOD, GST), cytoskeleton structure (actin) and embryo early development (vtg, initiation factor).

Detection of Zn2+ release in nitric oxide treated cells and proteome: dependence on fluorescent sensor and proteomic sulfhydryl groups.

Nitric oxide (NO) is both an important regulatory molecule in biological systems and a toxic xenobiotic. Its oxidation products react with sulfhydryl groups and either nitrosylate or oxidize them. The aerobic reaction of NO supplied by diethylamine NONOate (DEA-NO) with pig kidney LLC-PK1 cells and Zn-proteins within the isolated proteome was examined with three fluorescent zinc sensors, zinquin (ZQ), TSQ, and FluoZin-3 (FZ-3). Observations of Zn2+ labilization from Zn-proteins depended on the specific sensor used. Upon cellular exposure to DEA-NO, ZQ sequestered about 13% of the proteomic Zn2+ as Zn(ZQ)2 and additional Zn2+ as proteome·Zn-ZQ ternary complexes. TSQ, a sensor structurally related to ZQ with lower affinity for Zn2+, did not form Zn(TSQ)2. Instead, Zn2+ mobilized by DEA-NO was exclusively bound as proteome·Zn-TSQ adducts. Analogous reactions of proteome with ZQ or TSQ in vitro displayed qualitatively similar products. Titration of native proteome with Zn2+ in the presence of ZQ resulted in the sole formation of proteome·Zn-ZQ species. This result suggested that sulfhydryl groups are involved in non-specific proteomic binding of mobile Zn2+ and that the appearance of Zn(ZQ)2 after exposure of cells and proteome to DEA-NO resulted from a reduction in proteomic sulfhydryl ligands, favoring the formation of Zn(ZQ)2 instead of proteome·Zn-ZQ. With the third sensor, FluoZin-3, neither Zn-FZ-3 nor proteome·Zn-FZ-3 was detected during the reaction of proteome with DEA-NO. Instead, it reacted independently with DEA-NO with a modest enhancement of fluorescence.