[Show abstract][Hide abstract] ABSTRACT: Proteomics can be used to study the metabolic pathways and mechanisms involved in antimicrobial resistance. The aim of this comparative proteomic study was to establish the overall changes in the proteome of a naturally occurring ESBL-producing E. coli strain (C5478) stressed with its minimal inhibitory concentration (2 μg/mL) of cefotaxime, compared to the proteome of the same strain without antimicrobial stress, by using 2-D gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparative proteomic analysis revealed that the abundance of numerous protein species changed in the strain stressed by CTX compared to the non-stressed wild-type strain. A total of 188 spots were excised from the 2-DE gel of the wild-type strain, 112 of which were successfully identified by MALDI-TOF MS, representing 110 different proteins. Concerning the 2-DE gel of the CTX-stressed bacteria, 171 spots were excised and 156 were identified, representing 143 different proteins. The proteins identified in both strains were categorized according to their biological functions. These proteins were involved in metabolism, protein synthesis, cell division, stress responses, and antimicrobial resistance, among others. These findings will be helpful to further understand not only the antimicrobial resistance mechanisms, but also the role of wild animals as reservoirs in spreading antimicrobial-resistant bacteria into the environment.
[Show abstract][Hide abstract] ABSTRACT: The eutrophication of surface waters caused by cyanobacteria is a global problem, leading to increasing water treatment costs, a degraded recreational value, bad taste or odour and sometimes toxin production. The production of Microcystins, a Microcystis sp. Toxin, by these micro-algae may cause many health problems to humans and animals (e.g. liver cancer) and even death. A variety of methods have already been developed to control cyanobacteria blooms, including physical and chemical treatments. However, they have negative impacts on other species of algae and other aquatic organisms. Ultrasonic algae treatment has been proposed as a clean approach to controlling the blooms of some algae species and Microcystins degradation. Thus, ultrasonication known to cause diverse chemical, physical and biological effects have been proposed to
control algal bloom in eutrophic water bodies by disrupting the gas vesicles from cells, inhibiting photosynthesis, production of free radicals, destruction of membranes and consequently reducing the growth rate of algae . However, the degradation of Microcystins following ultrasonication is not consensual in current scientific literature and more studies are required. The current work studies the effects of ultrasonication on the degradation of Microcystins under a variety of conditions (changing frequency and time) by high performance liquid chromatography (HPLC) and Matrix-assisted laser desorption ionization time of flight (MALDI-TOF/ TOF MS).
[Show abstract][Hide abstract] ABSTRACT: DiAerent procedures for matrix assisted laser desorption ionization time of ;ight mass spectrometry-based
classi cation at the strain
level using the enzymatic digestion of proteins from the cell lysate have been studied. 2e eAects of ultrasonic energy as well as the eAects of
protein reduction and protein alkylation in the sample treatment and in the subsequent classi cation were assessed. 2e nal optimal meth-
od for classi cation was then compared with an intact cell-based approach in a diAerent set of samples. Our results show that
cation at the strain level is possible as 12 diAerent strains were correctly classi ed using intact cell analysis. Overall, the con dence level in
classi cation was higher when the analysis was performed with the intact cell approach.
[Show abstract][Hide abstract] ABSTRACT: The use of ultrasonic probe, in conjunction with immobilized trypsin, has been explored in this work for potential enhancement of protein digestion. Several solid supports commonly used to immobilize trypsin were subjected to different ultrasonication amplitudes and time in order to investigate their mechanical resistance to ultrasonic energy when provided by the ultrasonic probe. Glass beads and magnetic particles were found to remain intact in most conditions studied. It was found that immobilized trypsin cannot be reused after ultrasonication since the enzymatic activity was greatly diminished. For comparative purposes, vortex shaking was also explored for protein cleavage. Four standard proteins--bovine serum albumin, α-lactalbumin, carbonic anhydrase and ovalbumin--were successfully identified using peptide mass fingerprint, or peptide fragment fingerprint. In addition, the performance of the classical protein cleavage (overnight, 12 h) and the ultrasonic methods was found to be similar when the digestion of a complex proteome, human plasma, was assessed through 18-O quantification. The digestion yields found were 90-117% for the ultrasonic and 5-21% for the vortex when those methods were compared with the classical overnight digestion.
[Show abstract][Hide abstract] ABSTRACT: Herein we report results regarding the optimization and comparison between different ultrasonic-based procedures for protein quantitation by the direct (18) O-labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme-to-protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure.
Rapid Communications in Mass Spectrometry 01/2011; 25(1):75-87. DOI:10.1002/rcm.4816 · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new versatile emissive molecular probe (3) derived from 1,5-bis(2-aminophenoxy)-3-oxopenthane bearing two units of 6-nitro-4-oxo-4H-chromene-has been prepared by a Schiff-base condensation method using conventional and green, ultrasound-aided, methods. The dry yellow powder was char-acterized as the imine species (3). These imine species, however, where found to rapidly convert to their enamine form (4) in solution, under the presence of water traces. This reaction was computationally studied through Density Functional Theory (DFT) in order to investigate the relative stability of the molecular pair 3/4. The sensing properties of the enamine (4) towards various metal ions were in-vestigated via absorption and fluorometric titrations in solution in dichloromethane, acetonitrile and DMSO. The compound shows a fluorescent turn-off response in the presence of Cu 2þ , Zn 2þ , Cd 2þ , Hg 2þ and Ag þ over the other metal ions studied, such as Li þ , Na þ , K þ , Ca 2þ , Co 2þ and Ni 2þ , being stronger for Cu 2þ and Hg 2þ . The gas phase chemosensing abilities of (3) were also explored suggesting (3) as new active MALDI-TOF-MS matrix by two dry methods showing a strong selectivity towards Cu 2þ and Ag þ . Our preliminary results show promising uses of (3) supported in PPMA films as metal ion solid chemosensor.
[Show abstract][Hide abstract] ABSTRACT: In this paper we present two case studies of Proteomics applications development using the AIBench framework, a Java desktop application framework mainly focused in scientific software development. The applications presented in this work are Decision Peptide-Driven, for rapid and accurate protein quantification, and Bacterial Identification, for Tuberculosis biomarker search and diagnosis. Both tools work with mass spectrometry data, specifically with MALDI-TOF spectra, minimizing the time required to process and analyze the experimental data.
Journal of integrative bioinformatics 01/2011; 8(3):171. DOI:10.2390/biecoll-jib-2011-171
[Show abstract][Hide abstract] ABSTRACT: AIBench is a successful Java application framework focused in the biomedical field. In this paper, we demonstrate the suitability
of this framework in aiding the development of proteomics applications working with mass spectrometry data. We present two
case studies: Decision Peptide-Driven for accurate protein quantification and Bacterial Identification for Tuberculosis biomarker
search and diagnosis.
5th International Conference on Practical Applications of Computational Biology & Bioinformatics, PACBB 2011, April 6-8, 2011, Salamanca, Spain; 01/2011
[Show abstract][Hide abstract] ABSTRACT: Titanium dioxide (TiO2) nanoparticles (NPs) were among the first nanomaterials made readily commercially available. Nowadays these NPs are being used for a wide variety of industrial applications and consumer products, including disinfectant sprays, sunscreens and water treatment agents, which can lead to significant releases into the environment (Fig.1). Still, there is few information about the quantities that are being released and accumulated into the ecosystems. The lack of information about its potential toxicological effects and impact on the environment and Human health is a matter of great concern (Mendonça et al., 2010).
Bivalves have been recognized as sentinel organisms of aquatic ecosystems, since they provide information about the bioavailability and environmental impact of various compounds (Bilos et al., 1998). Corbicula fluminea (Muller, 1744), is a species of freshwater bivalves that lives on the sediment and that are filter feeders, effecting constant exchanges with the environment through a system of siphons (inhalant and exhalant) (Mouthon, 2001).
In this context, this study pretends to evaluate the toxicity caused by TiO2 NPs exposure to aquatic sediment-dwelling organisms, using as biological model freshwater bivalves (Corbicula fluminea).
2nd International Congress on Analytical Proteomics, ”. University of Vigo, Ourense, Spain, 18-20 July; 01/2011
[Show abstract][Hide abstract] ABSTRACT: Four strains of Listeria monocytogenes with different levels of virulence were studied. Two strains were consistently evaluated as virulent (strain 3077) and of low virulence (strain 3993), whereas the other two strains (3006 and 3049) originated conflicting results in what the evaluation tests were concerned: both were shown to exhibit low virulence when evaluated by in vitro assays, but virulent when the analyses were performed under in vivo conditions. To clarify the virulence potential of the selected strains, a proteomic approach was used after incubating L. monocytogenes cultures under conditions favoring the expression of virulence factors (minimal medium, at 37 °C). Bacterial proteins present in the liquid culture media were precipitated from late exponential phase cultures, fractionated by SDS-PAGE and identified by MALDI-TOF-MS. Three virulence factors differentially expressed were detected: protein p60, listeriolysin O (LLO) and internalin C (InlC). Clustering analysis of the four L. monocytogenes strains based on their secretome profiles allowed their categorization in two groups: the virulent group, composed by strains 3077 and 3049, and the low virulence group, containing strains 3993 and 3006. The results presented in this work suggest that the virulent potential of a particular L. monocytogenes strain may be predicted from the levels of both listeriolysin O (LLO) and internalin C (InlC) present in its secretome when the bacterium is grown under conditions favoring the expression of virulence factors. Following validation of this proposal through the analysis of a large array of strains, this methodology exhibits a great potential to be developed into an accurate and rapid method to characterize L. monocytogenes strain virulence.
[Show abstract][Hide abstract] ABSTRACT: We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on (18)O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant (18)O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that (18)O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.
[Show abstract][Hide abstract] ABSTRACT: We studied in this work the performance of the new ultrasonic multiprobe in terms of throughput, handling and robustness. The study was conducted using the multiprobe to speed two different proteomics workflows. The "classic" method relaying on overnight protein digestion (12h), was used as the standard procedure. This work clearly shows the importance of testing variables such as ultrasonic amplitude and ultrasonic time when adapting an ultrasonic-based treatment to a new ultrasonic device. The results here presented also shown and confirm the advantage of speed up sample treatment workflows with the aid of ultrasonic energy in combination with a 96-well plate. The methods compared were similar in terms of robustness, but the desalting free method was the fastest, requiring only 2 min/sample for completion. In addition it was also the simplest in terms of handling, since no desalting step was needed. The following standard proteins were successfully identified using the methods studied: bovine serum albumin, alpha-lactalbumin, ovalbumin, carbonic anhydrase, fructose-bisphosphate aldolase A, catalase, chymotrypsinogen A. As case study, the identification of the protein Split-Soret cytochrome c from D. desulfuricans ATCC 27774 was carried out.
[Show abstract][Hide abstract] ABSTRACT: Nowadays isotopic (18)O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using (18)O-labeling. In this critical review we comment on the different ways in which (18)O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage.
[Show abstract][Hide abstract] ABSTRACT: We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the (18)O-decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the (18)O-isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field.
[Show abstract][Hide abstract] ABSTRACT: Recent tools addressed to accelerate the different steps of the sample treatment for protein identification in modern workflows are reviewed and critically commented in this manuscript. Heating, microspin columns, ultrasonic energy, high pressure, infrared energy, microwave energy, alternating electric fields and microreactors are outlined as useful tools that can be used to accelerate all or some of the following steps for in-gel or in-liquid based approaches for protein identification: (i) protein dissolution/denaturation, (ii) protein reduction, (iii) protein alkylation and (iv) protein digestion. The advantages and drawbacks, along with the main differences among the different tools are also commented. Future prospects for hyphenation of methods are also discussed. Researchers are informed also in this work regarding the main problems to be found when implementing any of the above mentioned methods.
[Show abstract][Hide abstract] ABSTRACT: For microbial pathogens, secretory proteins are the frontline in host-pathogen interactions. The extracellular protein profiles of four strains of the foodborne pathogenic bacterium Listeria monocytogenes were compared in this study. These strains, belonging to four different serovars, were selected according to differences in virulence. In order to enhance the expression of the virulence factors, cultures of L. monocytogenes were incubated in minimal medium (Modified Welshimer Broth) at 37 ºC. Secreted proteins present in the supernatants of the cell cultures, in late exponential phase, were precipitated and separated by SDS-PAGE, using equivalent amounts of total secreted proteins in the wells (1). Differentially expressed peptides were identified by PMF and MALDI-TOF MS (2). The clustering of the four strains in three groups, based on proteomic patterns, was observed. Distribution of serovars in these clusters was in agreement with the proposed three L. monocytogenes genetic lineages (3). The intensities of two particular bands (in the range of 45 to 66 kDa), identified as virulence factors p60 and listeriolysin O, were not consistent among isolates. These results are in accordance with previous reports that used a more time consuming (2-D) electrophoresis approach (2). We present a simplified methodology to improve our knowledge on the secretome of different L. monocytogenes strains, aiming to develop more efficient strategies to knock out this pathogen in foods. Acknowledgments
5th Congress of the Portuguese Proteomics Network – PROCURA / 1st International Congress on Analytical Proteomics - ICAP, Caparica, Portugal; 09/2009
[Show abstract][Hide abstract] ABSTRACT: Preliminary results regarding fast isotopic labeling of proteins with (18)O in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry technique are presented. Similar (16)O/(18)O isotopic labeling ratios were found for the overnight procedure (12h) and the new fast ultrasonic one (30 min) for the BSA, ovalbumin and alpha-lactalbumin proteins. The procedure, however, failed to promote double (18)O isotopic labeling for the proteins, ovalbumin and alpha-lactalbumin. Two different sonication frequencies, 35 and 130 kHz, were studied at two different sonication times of 15 and 30 min, being best results obtained with the procedure at 130 kHz of sonication frequency and 30 min of sonication time. For comparative purposes the overnight isotopic (18)O labeling procedure was done. In addition, the new fast isotopic labeling procedure was also studied without ultrasonication, in a water bath at 60 degrees C.
[Show abstract][Hide abstract] ABSTRACT: An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate-reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774.
Rapid Communications in Mass Spectrometry 10/2007; 21(20):3269-78. DOI:10.1002/rcm.3214 · 2.25 Impact Factor