Anna Bodzon-Kulakowska

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.

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.

The Proteomic Analysis of Primary Cortical Astrocyte Cell Culture after Morphine Administration

Astrocytes are supportive cells, necessary for ensure optimal environment for neural cells functioning. They are involved in extracellular K+ level regulation and neurotransmitters removal. They are also dependent for myelination and synapses formation. They may make a contribution in signal propagation in the central nervous system, for example, through Ca2+ signaling. With the use of neonatal pure astrocyte cell culture, we investigated changes in astrocytes proteomes under the influence of morphine. We found 10 major proteins, which show different expression between physiological cell culture and morphine treatment. With 2D gel electrophoresis and nanoLC-ESI-MS/MS, we identified proteins and characterized their potential role in morphine dependence. Observed differences were also confirmed by Western blotting. Our data suggests a role for astrocytes in the formation of the morphine dependence at the molecular level. This finding may support interpretation of causes of morphine dependence formation based only on behavioral data.

Proteomic analysis of striatal neuronal cell cultures after morphine administration

Using primary neuronal cell culture assays, combined with 2-D gel electrophoresis and capillary LC-MS, we identified differences in proteomes between control and morphine-treated cells. Statistically significant differences were observed among 26 proteins. Nineteen of them were up-regulated, while seven were down-regulated in morphine-treated cell populations. The identified proteins belong to classes involved in energy metabolism, associated with oxidative stress, linked with protein biosynthesis, cytoskeletal ones, and chaperones. The detected proteins demand further detailed studies of their biological roles in morphine addiction. It is crucial to confirm observed processes in vivo in order to reveal the nature and importance of the biological effect of proteome changes after morphine administration. Further investigations may lead to the discovery of new proteome-based effects of morphine on living organisms.

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.

Morphinome – proteome of the nervous system after morphine treatment

Summary.Proteome is a natural consequence of the post-genome era when the HUGO project (Human Genome Organization) has almost been completed. Here, a specifically aimed proteome in drug dependence – morphinome, is described, including tasks, strategies and pitfalls of the methodology.

DESI–MS as a tool for direct lipid analysis in cultured cells

Desorption electrospray ionization may be used as a fast and convenient method for analysis and identification of lipids in the cell culture. Oxidative stress, which usually involves changes in lipids, was used as a model of pathology to show the utility of this analysis methodology. This paper addresses the surface preparation of cell culture slides, induction of oxidative stress, and cell monolayer culture preparation as well as optimization of the analysis. Advantages and drawbacks of the method were also discussed.

DESI analysis of mammalian cell cultures – sample preparation and method optimisation

Desorption electrospray ion source (DESI) is widely used as an MS imaging technique. It is a rapid and convenient method of surface analysis, but to date, there are methodological obstacles to its application to the analysis of cell culture. This study reported optimised conditions for the analysis of cell culture samples. Parameters such as the surface, medium removal and sample desiccation techniques were assessed as a function of output data quality. Supercharging agents, surfactants and optimal parameters for the DESI ion source were evaluated for use in cell culture analyses. Data indicated that plastic dishes or sodium glass coated with poly-l-lysine and washing cell cultures with 150 mM ammonium acetate followed by drying with inert gas were superior for DESI analyses. The addition of 1 μM surfactin to the DESI spray solvent significantly improved the results for negative and positive ion modes.

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

AbstractPeptide 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. FigureMonitoring of trypsin hydrolysis on PEG-peptide surface

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.