1,721,049 research outputs found
Development of molecular imprinted polymers selective for the enrichment of sulfopeptides in biological samples
No full textMagnetic materials for the selective analysis of peptide and protein biomarkers
No full textThis mini-review article provides an overview on the use of magnetic materials for the analysis of protein biomarkers. In particular, the advantage provided by magnetic solid phase extraction will be discussed with selected examples, considering untargeted analysis for screening new biomarker proteins and targeted investigation on known and suggested new biomarkers. Aspects, such as enrichment efficiency over conventional techniques, ease of use, functionalization versatility and automation will be considered, together with quantification and deeper structure elucidation provided by coupling selective or specific enrichment to powerful characterization techniques, such as mass spectrometr
Determination of Enantioselectivity and Enantiomeric Excess by Mass Spectrometry in the Absence of Chiral Chromatographic Separation: An Overview
No full textThis review describes the principles and instrumentation for the screening of asymmetric reactions by mass spectrometry. These techniques witnessed a significant advancement in the last few years. Although some of them are still at the proof-of-concept development stage, several applications might be foreseen in the field of combinatorial, high-throughput parallel catalyst screening
Detailed lipid investigation of edible seaweeds by photochemical derivatization and untargeted lipidomics
Full text linkSeaweeds are macrophytic algae that have been gaining interest as alternative healthy foods, renewable drug sources, and climate change mitigation agents. In terms of their nutritional value, seaweeds are renowned for their high content of biologically active polyunsaturated fatty acids. However, little is known about the regiochemistry-the geometry and position of carbon-carbon double bonds-of free and conjugated fatty acids in seaweeds. In the present work, a detailed characterization of the seaweed lipidome was achieved based on untargeted HRMS-based analysis and lipid derivatization with a photochemical aza-Patern & ograve;-B & uuml;chi reaction. A triple-data processing strategy was carried out to achieve high structural detail on the seaweed lipidome, i.e., (i) a first data processing workflow with all samples for aligning peak and statistical analysis that led to the definition of lipid sum compositions (e.g., phosphatidylglycerol (PG) 34:1), (ii) a second data processing workflow in which the samples of each seaweed were processed separately to annotate molecular lipids with known fatty acyl isomerism (e.g., PG 16:0_18:1), and (iii) the annotation of lipid regioisomers following MS/MS annotation of the lipid derivatives obtained following the aza-Patern & ograve;-B & uuml;chi reaction (e.g., PG 16:0_18:1 omega-9). Once the platform was set up, the lipid extracts from 8 seaweed species from different seaweed families were characterized, describing over 900 different lipid species, and information on the regiochemistry of carbon-carbon double bonds uncovered unknown peculiarities of seaweeds belonging to different families. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of seaweeds
Analytical strategies based on chromatography-mass spectrometry for the determination of estrogen-mimicking compounds in food
No full textFood safety can be compromised by the presence of a wide variety of substances, deriving from both natural and anthropogenic sources. Among these substances, compounds exhibiting various degrees of estrogenic activity have been widely studied in environmental samples, whereas less attention has been devoted to food matrices. The aim of the present review is to give a general overview on the recent analytical methods based on gas or liquid chromatography coupled to mass spectrometry for the determination of estrogen-like compounds in foods, including new developments, improvements and upcoming trends in the field. Attention will be focused on four representative groups of compounds, i.e. natural and synthetic estrogens, mycoestrogens, phytoestrogens, and alkylphenols. (c) 2013 Elsevier B.V. All rights reserved
Proteome characterization of platelet microparticles by nanoHPLC/high resolution mass spectrometry
No full textMicroparticles (MPs) were first described by Wolf in 1967 as a “platelet dust” present in blood [1], and since then they have drawn much attention. Now they are considered a heterogeneous vesicle population (100–1000 nm) virtually released by all eukaryotic cells in a highly controlled process triggered by various stimuli, such as cell activation after stimulation with proinflammatory, prothrombotic, or proapoptotic substances, stress conditions, but also cellular differentiation, senescence, apoptosis and cell damage. It is supposed that MPs formation affects their features phenotypically and quantitatively, also for the same type of cell [2].
MP release is a fundamental capacity because it allows cells to selectively concentrate and release part of their content into the surrounding milieu [2]. For this reason MPs still retain part of the proteins originally belonging to the parental cell [3]. Evidence has showed their involvement in the local and systemic intracellular communication by two mechanisms. MPs can behave as circulating messengers and interact with target cells exposing membrane-associated, bioactive molecules; otherwise, MPs can act as vehicles and directly transfer part of their content, in particular proteins, RNA and bioactive lipids, inducing activation, phenotypic modifications or reprogramming in the target cell. Such processes are involved in HIV spreading, atherosclerosis, vascular regeneration and propagation of oncogenic activity. The presence of negatively charged phospholipids promotes the formation of procoagulant protein complexes, both in disease and healthy states, contributing to haemostasis [4].
MPs can be found in blood of both healthy and diseased individuals; however, the number of circulating MPs, cellular origin and composition vary according to type and state of a disease and medical treatment. Huge levels of MPs are generally associated with thrombotic tendencies and an increase of MPs has been documented for several pathological conditions, such as atherothrombosis, thrombocytopenia, ischemic attacks, cardio vascular disease but also for diabetes, sepsis, cancer and autoimmune diseases [4,5]. On the contrary, a decrease in MPs concentration has been documented for bleeding disorders [5].
Despite their important and interesting biological roles, currently there is no standardized method for qualitative and quantitative analysis of MPs, and several approaches have been described, such as flow cytometry, electron microscopy, ELISA and proteomic methods [5].
Flow cytometry is a traditional method for PMs counting and it also allows the determination of cell markers, but it cannot distinguish MPs from other small objects, such as cell debris or aggregates, and MP size is close to the inferior limit of the instrument, with subsequent loss of accuracy. Proteomics is a more powerful and widely used tool for MP study, but most importantly for their characterization. Most of the proteomic studies exploit a general workflow, in which MPs are isolated by differential centrifugation and then the protein content is quantified by a classical assay; finally proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in-gel trypsin digested and identified by a mass spectrometric technique. SDS-PAGE has the advantage of being able to separate both membrane and soluble proteins with good compatibility to mass spectrometric analysis; however, two dimension-PAGE is also possible and provides a better protein separation, although less suitable for the analysis of hydrophobic proteins [6]. Both methodologies enhance protein identification but they are operatively complex and time-consuming, thus not suitable for high-throughput analyses.
In this context, we chose to apply a modern shotgun proteomics approach already successfully used for the characterization of several other complex biological samples; the aim of this project is to provide a simplified and better experimental procedure for the characterization of platelet microparticles (PMPs), in order to furnish the analytical tools for protein identification and the basis for understanding their roles in cell communication.
PMPs have been isolated from ADP-stimulated platelets, which in turn were obtained from the blood of healthy volunteers by differential centrifugation, at low speed to remove red blood cells and leukocytes, and at higher speed to sediment platelets. Incubation with ADP to induce the release of PMPs has been chosen according to previous works [3], because it is an endogenous physiologically relevant compound which can activate platelets in few minutes. PMPs were then sedimented by ultracentrifugation for 1 hour and solubilized in a SDS solution, a commonly used surfactant for cell lysis, to break their structure and release proteins. The obtained proteins were quantified by the standard Bradford assay to determine the amount that should be further processed.
At this point samples were split into two aliquots and processed according to two different procedures. One was analyzed by standard shotgun proteomics, thus denaturated and tryptic digested. The other aliquot was fractionated using a modern technology, hydrogel nanoparticles, a prefractionation system successfully used for different biological matrices analysis to improve protein identification of low molecular weight proteins. This was chosen to tackle a common issue with shotgun approaches, in which the low-abundance proteins (mainly with regulatory functions) are not detected when surveys are on a broad scale, thus focused on high-abundance proteins, with subsequent suppression of signals of low-abundance peptides during MS acquisition. After hydrogel nanoparticle procedure, eluted proteins were quantified by Bradford assay to choose the proper amount of sample that would be subsequently in-solution digested.
Both standard and hydrogel nanoparticle processed samples were off-line desalted using C18-columns and analyzed. All peptide mixture were injected into a reversed-phase nanoHPLC-LTQ Orbitrap XL mass spectrometer system. The experiment was performed in triplicate and three technical replicates (nanoHPLC-MS/MS runs) were performed for each sample. Raw data were then analyzed to retrieve the proteins present in the sample by means of Mascot database search and Scaffold validation. With this approach more than 500 proteins were validated, and among them more than 200 proteins were never found in previous PMP studies. In conclusion, we provided a more simple and straightforward procedure for the study of PMPs, producing a tool for further understanding their biological and pathological roles. These results meet the criteria of excellent science and better society in Horizon 2020 framework program for research and innovation
Membrane proteome functional characterization of breast cancer-initiating cells subjected to bone morphogenetic protein signaling inhibition by dorsomorphin
No full textIn this study, A17 cells, which are an invasive mesenchymal cell line with cancer stem cell properties, were exploited for the study of the role of bone morphogenetic protein pathways in cancer-initiating cells employing a proteomics-based approach. A17 cells were treated with the bone morphogenetic protein signaling inhibitor dorsomorphin for 3 days. After that, subcellular fractionation of cell samples was performed and the membrane fraction analyzed by shotgun proteomics. The extracted membrane proteins were enzymatically digested and the resulting peptide mixture was analyzed by nano liquid chromatography coupled to tandem mass spectrometry and relative label-free quantitation. Protein profiles of A17 membrane fractions before and after dorsomorphin treatment were compared, and further mined by Gene Ontology search. The protein profile of untreated A17 samples correlated with the mesenchymal phenotype, whereas changes were observed in dorsomorphin-treated samples, further supporting a mesenchymal to epithelial transition upon bone morphogenetic protein signaling pathway inhibition and the importance of this pathway in breast cancer cell malignancy
Analytical Methods for Characterizing the Nanoparticle-Protein Corona
No full textWhen nanoparticles (NPs) enter a biological environment, medium components, especially proteins, compete for binding to the NP's surface, leading to development of a new interface, commonly referred to as the "protein corona." This rich protein shell gives the NPs a biological identity that can be very different from their synthetic one, in terms of their chemical-physical properties. Understanding NP-protein interaction is crucial for both the bioapplications and safety of nanomaterials. The protein corona provides the primary contact to the cells and their receptors. It defines in vivo fate of the delivery systems, governing the stability, immunogenicity, circulation, clearance rates and organ biodistribution of the NPs. Given its importance, the application and the development of analytical methods to investigate the protein corona are crucial. This review gives an overview of chromatographic, electrophoretic, mass spectrometric and proteomic methods because these techniques have the advantage to be able to identify and quantify individual proteins adsorbed onto the corona. This capability opens up the possibility to exploit the protein corona for specific cell targeting. © 2014 Springer-Verlag
Proteome investigation of the non-model plant pomegranate (Punica granatum L.)
No full textA gel-free, shotgun proteomics approach was used to characterize pomegranate aril proteome by nanoliquid chromatography-high-resolution tandem mass spectrometry. To identify both high-abundance and low-abundance proteins, we applied two distinct sample preparation protocols, i.e., a classical one widely applied in literature and a second one able to reduce the dynamic range of protein concentration of the sample, based on combinatorial hexapeptide ligand library technology. However, the proteins identified with the latter protocol were only a small minority. Because pomegranate is a non-model plant species, i.e., information of its genome sequence are lacking, only a few protein sequences are included in the most widely known protein sequence databases. To improve both the number of identified proteins and data reliability, identification was performed integrating the results obtained with three distinct plant protein databases, since the majority of proteins could only be attributed by homology with other plant species. Nevertheless, many proteins had assigned only one unique peptide, because of the phylogenetic distance of pomegranate from the main model plants. After manual revision of the identified proteins to eliminate the redundant or ambiguous identifications, a list of 1,488 proteins was obtained, only six of which belonging to pomegranate species. To the author's best knowledge, this is the first work aimed at the proteomic characterization of Punica granatum
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