Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

Matrix-assisted laser desorption/ionization mass spectrometry imaging sample preparation using wet-interface matrix deposition for lipid analysis.

RATIONALE: Sample preparation is one of the most crucial steps for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Scientists beginning their study with this technique may be overwhelmed by the variety of matrices, solvents, and concentrations; the methods of their applications; and the lack of widely available knowledge of the effect of these parameters on the results. Here we present in depth the aspects of matrix deposition, which will be helpful for the scientific community. METHODS: In this study, we tested several MALDI matrices, such as 2,5-dihydroxybenzoic acid (DHB), norharmane, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), and 9-aminoacridine (9AA), using the SunCollect system: wet-interface matrix deposition in the context of lipid analysis. We optimized the number of matrix layers and nozzle settings in terms of spectral intensity and the overall quality of the obtained ion maps. RESULTS: Our research presents the effect of the number of matrix layers and nozzle settings on the results and allows for choosing the optimal parameters for the analyses. In positive ionization mode, DHB matrix could be chosen first. In the negative ionization mode, 1,5-diaminonaphthalene matrix produces a higher peak intensity in a lower mass range and seems to provide more information than 9AA. We recommend NEDC for particular processes such as glucose analysis. Compared to the remaining matrices, norharmane shows significant changes in the obtained ion maps. CONCLUSIONS: Such a large amount of data allow us to observe an interesting conclusion: the obtained ion image for a particular ion could differ dramatically with a change in the matrix, the solvent composition, or even the number of matrix layers. This must be considered when interpreting the result, impelling us to compare the results obtained with different matrices with caution.

MASS SPECTROMETRY IN VIROLOGICAL SCIENCES.

Virology, as a branch of the life sciences, discovered mass spectrometry (MS) to be the pivotal tool around two decades ago. The technique unveiled the complex network of interactions between the living world of pro- and eukaryotes and viruses, which delivered "a piece of bad news wrapped in protein" as defined by Peter Medawar, Nobel Prize Laureate, in 1960. However, MS is constantly evolving, and novel approaches allow for a better understanding of interactions in this micro- and nanoworld. Currently, we can investigate the interplay between the virus and the cell by analyzing proteomes, interactomes, virus-cell interactions, and search for the compounds that build viral structures. In addition, by using MS, it is possible to look at the cell from the broader perspective and determine the role of viral infection on the scale of the organism, for example, monitoring the crosstalk between infected tissues and the immune system. In such a way, MS became one of the major tools for the modern virology, allowing us to see the infection in the context of the whole cell or the organism. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

The Study of Derivatization Prior MALDI MSI Analysis-Charge Tagging Based on the Cholesterol and Betaine Aldehyde.

Mass spectrometry imaging is a powerful tool for analyzing the different kinds of molecules in tissue sections, but some substances cannot be measured easily, due to their physicochemical properties. In such cases, chemical derivatization could be applied to introduce the charge into the molecule and facilitate its detection. Here, we study cholesterol derivatization with betaine aldehyde from tissue slices and evaluate how different sample preparation methods influence the signal from the derivatization product. In this study, we have tested different solutions for betaine aldehyde, different approaches to betaine aldehyde deposition (number of layers, deposition nozzle height), and different MALDI matrices for its analysis. As a result, we proved that the proposed approach could be used for the analysis of cholesterol in different tissues.

Coacervate Thermoresponsive Polysaccharide Nanoparticles as Delivery System for Piroxicam.

Low water solubility frequently compromises the therapeutic efficacy of drugs and other biologically active molecules. Here, we report on coacervate polysaccharide nanoparticles (CPNs) that can transport and release a model hydrophobic drug, piroxicam, to the cells in response to changes in temperature. The proposed, temperature-responsive drug delivery system is based on ionic derivatives of natural polysaccharides-curdlan and hydroxypropyl cellulose. Curdlan was modified with trimethylammonium groups, while the anionic derivative of hydroxypropyl cellulose was obtained by the introduction of styrenesulfonate groups. Thermally responsive nanoparticles of spherical shape and average hydrodynamic diameter in the range of 250-300 nm were spontaneously formed in water from the obtained ionic polysaccharides as a result of the coacervation process. Their morphology was visualized using SEM and AFM. The size and the surface charge of the obtained objects could be tailored by adjusting the polycation/polyanion ratio. Piroxicam (PIX) was effectively entrapped inside the nanoparticles. The release profile of the drug from the CPNs-PIX was found to be temperature-dependent in the range relevant for biomedical applications.

Glycosylation Changes in Serum Proteins Identify Patients with Pancreatic Cancer.

After more than a decade of biomarker discovery using advanced proteomic and genomic approaches, very few biomarkers have been involved in clinical diagnostics. Most candidate biomarkers are focused on the protein component. Targeting post-translational modifications (PTMs) in combination with protein sequences will provide superior diagnostic information with regards to sensitivity and specificity. Glycosylation is one of the most common and functionally important PTMs. It plays a central role in many biological processes, including protein folding, host-pathogen interactions, immune response, and inflammation. Cancer-associated aberrant glycosylation has been identified in various types of cancer. Expression of cancer-specific glycan epitopes represents an excellent opportunity for diagnostics and potentially specific detection of tumors. Here, we report four proteins (LIFR, CE350, VP13A, HPT) found in sera from pancreatic cancer patients carrying aberrant glycan structures as compared to those of controls.

Proteomic Data in Morphine Addiction Versus Real Protein Activity: Metabolic Enzymes.

Drug dependence is an escalating problem worldwide and many efforts are being made to understand the molecular basis of addiction. The morphine model is widely used in these investigations. To date, at least 29 studies exploring the influence of morphine on mammals' proteomes have been published. Among various proteins indicated as up- or down-regulated, the expression changes of enzymes engaged in energy metabolism pathways have often been confirmed. To verify whether proteomics-indicated alterations in enzyme levels reflect changes in their activity, four enzymes: PK, MDH, Complex I, and Complex V were investigated in morphine addiction and abstinence models. After analyses of the rat brain mitochondria fraction in the model of morphine dependence, we found that one of the investigated enzymes (pyruvate kinase) showed statistically significant differences observed between morphine, control, and abstinence groups. J. Cell. Biochem. 118: 4323-4330, 2017. © 2017 Wiley Periodicals, Inc.

Brain lipidomic changes after morphine, cocaine and amphetamine administration - DESI - MS imaging study.

Drug addiction is a complex disorder, evoking significant changes in the proteome of the central nervous system. To check if there are also changes in the lipidomic profiles we used desorption electrospray-MS technique for imaging of the brain slices of rats exposed to morphine, cocaine and amphetamine. Our investigations showed alternative regulation of selected lipid's levels in the central nervous system structures, under the influence of applied drugs. Results of our investigations can show changes in the brain treated with drugs of abuse in the new light, indicating role of the lipids in the addiction development.

Imaging mass spectrometry: Instrumentation, applications, and combination with other visualization techniques.

Imaging Mass Spectrometry (IMS) is strengthening its position as a valuable analytical tool. It has unique ability to identify structures and to unravel molecular changes that occur in the precisely defined part of the sample. These unique features open new possibilities in the field of various aspects of biological research. In this review we briefly discuss the main imaging mass spectrometry techniques, as well as the nature of biological samples and molecules, which might be analyzed by such methodology. Moreover, a novel approach, where different analytical techniques might be combined with the results of IMS study, is emphasized and discussed. With such a fast development of IMS and related methods, we can foresee the promising future of this technique.

Protocol: MYTHBUSTERS: a universal procedure for sample preparation for mass spectrometry.

Improvements in proteomic strategies from the development of new and robust separation and identification techniques have led to broad applications of proteomics to solve numerous biological questions. For all analyses, sample quality is unquestionably a critical factor; therefore protein extraction is of outmost importance. The ideal extraction method should provide reproducible spectra of the most comprehensive repertoire of proteins, while minimizing sample loss and degradation. It is already known that to capture the whole proteome is an unenforceable task. Many protein extraction protocols have been described, yet there is no "one perfect procedure" taking into account the vast diversity of biological and physical properties of proteins, including their charge, size, hydrophobicity, interactions and sub-cellular localization. The research presented here reflects the main obstacle occurring in proteomic experimental design; i.e. the lack of reproducibility as a result of alterations in protein extraction methods. We have performed a series of experiments, aimed towards identification of the aptamer-binding partners in cancerous cells. Aptamers are chemically synthesized, short, single-stranded nucleic acids with a strictly defined three-dimensional structure, which allows them to interact with a target molecule with high affinity. The low immunogenicity and cellular- targeting properties of aptamers might facilitate design of suitable drugs with low side-effects. Aptamers can be used for identification of molecules associated with a pathogenic state of a cell. Aptamers can be considered as a powerful tool, since they possess unique properties to benefit cancer diagnosis, prevention and treatment. We have used different types of protein extraction methods prior to analyses of complex biological samples by mass spectrometry, based on slight changes of homogenization buffers, and have observed the changes in the identified compounds. These results should prove to be very useful for future proteomic studies and the design of studies in terms of sample preparation, especially sample homogenization and protein extraction.

Comparison of two freely available software packages for mass spectrometry imaging data analysis using brains from morphine addicted rats.

Data analysis from mass spectrometry imaging (MSI) imaging experiments is a very complex task. Most of the software packages devoted to this purpose are designed by the mass spectrometer manufacturers and, thus, are not freely available. Laboratories developing their own MS-imaging sources usually do not have access to the commercial software, and they must rely on the freely available programs. The most recognized ones are BioMap, developed by Novartis under Interactive Data Language (IDL), and Datacube, developed by the Dutch Foundation for Fundamental Research of Matter (FOM-Amolf). These two systems were used here for the analysis of images received from rat brain tissues subjected to morphine influence and their capabilities were compared in terms of ease of use and the quality of obtained results.

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines.

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6-18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.

Desorption electrospray ionisation (DESI) for beginners--how to adjust settings for tissue imaging.

RATIONALE: Desorption electrospray ionisation (DESI) is the ambient technique used for surface imaging. Despite its simplicity, the proper use of this technique is not easy, and usually leads to discouragement, especially in the case of biological sample measurements. Here, we present some tips and tricks which may be helpful during a complex process of ion source optimisation to achieve the desired results. METHODS: Rat liver tissue as an example of a biological sample and a surface covered with rhodamine-containing marker were measured using a DESI ion source (OMNIspray source, Prosolia, Indianapolis, IN, USA) connected to a AmaZon ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). RESULTS: The geometry of the ion source (nebulisation capillary angle, its distance to the surface, and to the MS inlet), and other settings like nebulising gas pressure, solvent flow and capillary voltage, were changed during the optimisation process. The results obtained for different parameters are presented. CONCLUSIONS: Differences between the results and the method of optimisation for biological and non-biological samples were shown. The influence of different parameters on the quality of mass spectra was indicated. Optimal parameters for the tissue and non-biological sample analysis were suggested.

iTRAQ analysis with Paul ion trap-obstacle solved.

Mass spectrometers equipped with ion trap analyzers have been significantly improved due to their high performance and wide application area accompanying the low costs of purchase. Despite several advantages, such as reasonable resolution at low cost, high sensitivity, and capability for multistage analysis, ion traps have an important drawback: low mass cutoff during tandem mass spectrometry analysis MS(n). Although the low mass cutoff associated with the ion trap does not seriously obstruct peptide identification, it may cause a serious problem in identification of small molecules (posttranslational modifications, e.g., glycan structures) and quantification of peptides with multiplexed isobaric tag reagents. The presented approach offers the possibility to use isobaric tags for relative and absolute quantification labeling (iTRAQ) for quantitative, proteomic analysis using typical, widely available ion trap devices and manufacturer's software. We have performed series of analyses of standard protein labeled with isobaric tags in various concentration ratios to prove quantitative capabilities of this approach.

Synthesis and characterisation of PEG-peptide surfaces for proteolytic enzyme detection.

Peptide surfaces were obtained by the covalent immobilisation of fluorescently labelled pentapeptides carboxyfluorescein-glycine-arginine-methionine-leucine-glycine, either directly or through a poly(ethylene glycol) (PEG) linker on modified silicon wafers. Each step during the preparation of the peptide surfaces was confirmed by several surface characterisation techniques. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy were used to determine the surface composition, the wafers philicity was measured by contact angle and atomic force microscopy was used to investigate the surface morphology. Exposure of the peptide surfaces to trypsin resulted in the release of a fluorescently labelled peptide product, which allowed the kinetics of the enzymatic reaction to be followed with the aid of fluorescence spectroscopy. The electrospray ionisation mass spectrometry analysis of the post-digestion solution confirmed that the pentapeptides attached to the solid support undergo specific trypsin hydrolysis at the C-terminus of the arginine residues. Detailed surface analyses before and after the enzyme action was performed using ToF-SIMS. Because of the limited accessibility of the short peptide directly attached to the surface, a quantitative yield of enzymatic hydrolysis was observed only in case when the peptide was bound through the PEG linker. The insertion of the PEG linker increased the number of immobilised peptides and the rate of enzymatic digestion which consequently improved the quality of the enzyme assays. The described approach may be used for different peptide sequences designed for other proteases.

The influence of matrix concentration and solvent composition on the results of MALDI MSI, with the aid of wet-interface matrix deposition.

Imaging mass spectrometry is a powerful technique for the molecular analysis of tissue sections. As in many analytical methods, sample preparation is one of the main and most important steps to obtain results of good quality. Usually, the matrix concentration and solvent composition in different studies are taken for granted without any further consideration. In our studies, we aimed to find how matrix concentration and a type of solvent influence the signal. Moreover, we also aimed to find the relationship between these parameters, how they influence the spectra, and how they influence obtained ion maps. In our experiments, we used SunCollect®, which is a commercially available wet-interface system for matrix deposition. We decided to choose two matrix concentrations (2,5-dihydroxybenzoic acid [DHB]: 15 and 25 mg/mL; 9-aminoacridine [9AA]: 7 and 5 mg/mL) and two different water solutions of solvents in two different percentages for the matrices (DHB: 50% and 70% of methanol [MeOH] and acetonitrile [ACN]; 9AA 70% and 50% of ethanol [EtOH] and MeOH). In the end, the influence of these parameters on obtained spectra and ion maps was assessed.

Mammalian Oocyte Analysis by MALDI MSI with Wet-Interface Matrix Deposition Technique.

Oocytes are a special kind of biological material. Here, the individual variability of a single cell is important. It means that the opportunity to obtain information about the lipid content from the analysis of a single cell is significant. In our study, we present a method for lipid analysis based on the MALDI-based mass spectrometry imaging (MSI) approach. Our attention was paid to the sample preparation optimization with the aid of a wet-interface matrix deposition system (matrix spraying). Technical considerations of the sample preparation process, such as the number of matrix layers and the position of the spraying nozzle during the matrix deposition, are presented in the article. Additionally, we checked if changing the 2,5-dihydroxybenzoic acid (DHB) and 9-Aminoacridine (9AA) matrix concentration and their solvent composition may improve the analysis. Moreover, the comparison of paraformaldehyde-fixed versus nonfixed cell analysis was performed. We hope that our approach will be helpful for those working on lipid analyses in extraordinary material such as a single oocyte. Our study may also offer clues for anybody interested in single-cell analysis with the aid of MALDI mass spectrometry imaging and the wet-interface matrix deposition method.

Mass Spectrometry Imaging of Biomaterials.

The science related to biomaterials and tissue engineering accounts for a growing part of our knowledge. Surface modifications of biomaterials, their performance in vitro, and the interaction between them and surrounding tissues are gaining more and more attention. It is because we are interested in finding sophisticated materials that help us to treat or mitigate different disorders. Therefore, efficient methods for surface analysis are needed. Several methods are routinely applied to characterize the physical and chemical properties of the biomaterial surface. Mass Spectrometry Imaging (MSI) techniques are able to measure the information about molecular composition simultaneously from biomaterial and adjacent tissue. That is why it can answer the questions connected with biomaterial characteristics and their biological influence. Moreover, this kind of analysis does not demand any antibodies or dyes that may influence the studied items. It means that we can correlate surface chemistry with a biological response without any modification that could distort the image. In our review, we presented examples of biomaterials analyzed by MSI techniques to indicate the utility of SIMS, MALDI, and DESI-three major ones in the field of biomaterials applications. Examples include biomaterials used to treat vascular system diseases, bone implants with the effects of implanted material on adjacent tissues, nanofibers and membranes monitored by mass spectrometry-related techniques, analyses of drug-eluting long-acting parenteral (LAPs) implants and microspheres where MSI serves as a quality control system.

Morphinome--a meta-analysis applied to proteomics studies in morphine dependence.

This review is a meta-analysis of data describing proteins regulated by morphine influence studied worldwide across last years administration. Up to date (July 2010), 15 studies concerning this subject have been published. Animal models, examined brain structures, the route of morphine administration and proteomic platforms used for identification of differentially expressed proteins were described. Standardization of obtained results allowed for creation of database of proteins, whose expression was altered by morphine administration (www.addiction-proteomics.org). Their analysis by tools available in Celera Panther Database was possible too. Proteins, which seem to be the most promising candidates for further research, due to their consistent appearance in different studies, were indicated. Created database may facilitate further studies by providing a possibility to compare results obtained during different experiments. At the end, dynamic picture of proteome after morphine administration, which emerges from the obtained results, is discussed and need for standardization of proteomics experiments is stressed. As meta-analysis is a very powerful tool for evaluation and comparison of multiple data. We believe this approach will be useful in the nearest future to extract vital information from a vast number of similar publications. Morphinome database created already by our group is a comfortable tool for validation and verification of new data received after morphine influence on proteomes investigations. It gives a chance for fast comparison of results without hours spent on life science literature mining.

Rapamycin Improves Spatial Learning Deficits, Vulnerability to Alcohol Addiction and Altered Expression of the GluN2B Subunit of the NMDA Receptor in Adult Rats Exposed to Ethanol during the Neonatal Period.

Ethanol exposure during pregnancy alters the mammalian target of rapamycin (mTOR) signaling pathway in the fetal brain. Hence, in adult rats exposed to ethanol during the neonatal period, we investigated the influence of rapamycin, an mTOR Complex 1 (mTORC1) inhibitor, on deficits in spatial memory and reversal learning in the Barnes maze task, as well as the ethanol-induced rewarding effects (1.0 or 1.5 g/kg) using the conditioning place preference (CPP) paradigm. Rapamycin (3 and 10 mg/kg) was given before intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4-9 (an equivalent to the third trimester of human pregnancy). Spatial memory/reversal learning and rewarding ethanol effect were evaluated in adult (PND60-70) rats. Additionally, the impact of rapamycin pre-treatment on the expression of the GluN2B subunit of NMDA receptor in the brain was assessed in adult rats. Our results show that neonatal ethanol exposure induced deficits in spatial memory and reversal learning in adulthood, but the reversal learning outcome may have been due to spatial learning impairments rather than cognitive flexibility impairments. Furthermore, in adulthood the ethanol treated rats were also more sensitive to the rewarding effect of ethanol than the control group. Rapamycin prevented the neonatal effect of ethanol and normalized the GluN2B down-regulation in the hippocampus and the prefrontal cortex, as well as normalized this subunit's up-regulation in the striatum of adult rats. Our results suggest that rapamycin and related drugs may hold promise as a preventive therapy for fetal alcohol spectrum disorders.