Arabidopsis thaliana, Hordeum vulgare, genus Lupinus, Medicago truncatula, metabolomics, primary and secondary metabolites, metabolites profiling, natural products, proteomics, biomarkers of human diseases, blood serum, biotic and abiotic stress, mass spectrometry, analysis of compositions, GC/MS techniques, LC/MS, CID MS/MS, structural analysis, plant immune system, flavonoids, glucosinolates, tryptophan derivatives, glycosides, cell wall
The studies of the Laboratory of Proteomics and Metabolomics include research on plant and animal systems and are based on proteomic and metabolomic analysis. The latter approach specifically applies the methods of primary and secondary metabolite profiling with the use of chromatographic separation techniques and identification and/or quantitative analysis of compounds in complex extracts by various detection systems (i.e., mass spectrometry, fluorescence and UV detectors). The research work is connected with the analysis of the role and importance of primary and secondary metabolites in fitness mechanisms of selectedplant species (A. thaliana, M. truncatula, H. vulgare, Lupinus spp.)exposed to different types of biotic and abiotic stress. In case of the model system that is based on A. thalianaplants, an attempt is being made to determine the functions of particular genes that participate in the synthesis of indole-type secondary metabolites. This goal is addressed by phenotypic analysis of mutant lines that are defective in expression of selected candidate genes.
Except from monitoring the biochemical processes in planta, the research work focuses on the application of various mass spectrometry techniques to determine the structure and identify natural products that occur in biological material, mostly the substances that are isomers or isobars. For that purpose, we use mass spectrometers with high-resolution analyzers (that usually measure ion flight time) and collision induced dissociation techniques (CID MS/MS) applying soft ionization methods (electro spray ionization).
An important research direction is constituted by projects that involve studies on proteins of both plant and animal origin. The first group of these projects is devoted to the proteomic analysis of barley seedlings exposed to drought stress. This research is carried out on various varieties and mapping populations of barley. The other group of proteomic studies involves application of proteomic analysis in medical diagnostics. These projects involve studies on labeling of human disease biomarkers at protein level or the analysis of the level of their post-translational modifications. For example, we use mass spectrometry techniques to identify the level of N-homocysteinylation in human proteins. Furthermore, our studies are devoted to carcinogenesis and abnormalities of the cardio-vascular system, particularly arteriosclerosis. In this specific case, we identify the modifications (changes) in blood proteins and elaborate methods for their quantitative labeling. The medical applications projects are carried out in cooperation with the Institute of Oncology Gliwice Branch, Faculties of Medicine in Poznań and Wrocław, and the University of New Jersey Medical School.
Current research activity
- The role of phenolic secondary metabolites in lupine (L. angustifolius) seedling defense against the anthracnose fungus Colletotrichum lupini and the participation of ABC transporters in transport of metabolites.
- Application of high resolution mass spectrometry techniques with collision induced dissociation (CID MS/MS) for the description of secondary metabolites that occur in compositions.
- The use of Maldi ToF mass spectrometer to monitor changes in the composition of selected secondary metabolites of biological material samples without a necessity to apply chromatography methods, a fingerprint approach for selected classes of natural products.
- Characterization of indole glucosino late metabolic pathway in Arabidopsis and its function in resistance to infection.
- Tryptophan derivatives in defense responses of Brassicaceae plants.
- The analysis of plant proteomes in barley seedlings of mapping varieties and populations exposed to drought stress with regard to the stress resistance of the selected lines.
- A search for human disease biomarkers in blood serum/plasma, using the 2DE/MS methods.
- The use of mass spectrometry to analyze protein n-homocysteinylation.
Most important research achievements
- We developed methods to apply the LC/MS/MS systems for structural characterization of secondary metabolites that occur in complex extracts derived from biological material.
- We identified protein serum amyloid (SAA) to differentiate blood serum sample of the lung cancer patients from healthy individuals. The research work was conducted in cooperation with the Department of Experimental and Clinical Radiobiology, Maria Sklodowska Curie Memorial Cancer Centre and Institute of Oncology, Gliwice Branch.
- We identified ?-1-microglubulin as a biomarker of kidney dysfunction with better sensitivity than the usually used diagnostic markers. Accumulation of the protein shows linear correlation with the progress of chronic kidney disease; furthermore, it allows for early detection of kidney dysfunction. Also, we identified the proteins that indicate different mechanism of atherosclerotic plaque formation in typical atherosclerosis and kidney disease-related atherosclerosis.
Actual research projects
- Correlation between the gene expression level of phenylopropanoid pathways and phenolic profile of secondary metabolites in the seedlings of narrowleaf lupine varieties and lines in the assessment of resistance to fungal pathogen infection (National Science Centre).
- The use of tandem mass spectrometry with collision induced dissociation for the profiling and analysis of phenolic secondary metabolites in multicomponent extracts of plant material (National Science Centre).
- The analysis of N-homocysteinylation of human plasma fibrinogen with chemical methods and mass spectrometry methods with regard to cardiovascular diseases (National Science Centre).
- Biotechnological tools to obtain drought-resistant crops. POLAPGEN-BD (Information Processing Institute ? structural funds 01.03.01).
- Functional characterization of pathogen inducible secondary metabolites in model plants (EMBO ? Installation Grant for the years 2011-2013).
P. Kachlicki, J. Einhorn, D. Muth, L. Kerhoas, M. Stobiecki
Evaluation of glycosylation and malonylation patterns in flavonoid glycosides during LC/MS/MS metabolite profiling.
Journal of Mass Spectrometry 43, 572-586 (2008).
D. Muth, E. Marsden-Edwards, P. Kachlicki, M. Stobiecki
Differentiation of Isomeric Malonylated Flavonoid Glyconjugates in Plant Extracts with UPLC-ESI/MS/MS
Phytochemical Analysis. 19, 444?452 (2008).
M. Pietrowska, Ł. Marczak, J. Polanska, K. Behrendt, E. Nowicka, A. Walaszczyk, A. Chmura, R. Deja, M. Stobiecki, A. Polanski, R. Tarnawski, P. Widlak
Mass spectrometry-based serum proteome pattern analysis in molecular diagnostics of early stage breast cancer.
Journal of Translational Medicine 7, 60 (2009).
Ł. Marczak, M. Stobiecki, M. Jasiński, W. Oleszek, P. Kachlicki
Fragmentation Pathways of Acylated Flavonoid Diglucuronides from Leaves of Medicago truncatula.
Phytochemical Analysis 21, 224-233 (2010)
M. Sikora, Ł. Marczak, T. Twardowski, M. Stobiecki, H. Jakubowski
Direct monitoring of albumin lysine-525 N homocysteinylation in human serum by liquid chromatography/mass spectrometry.
Analytical Biochemistry 405, 132-134 (2010).
B. Swarcewicz, A. Staszków, J. Banasiak, D. Muth, M. Jasiński, M. Stobiecki
LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula.
Metabolomics 7, 604?613 (2011).
Ł. Marczak, M. Sikora, M. Stobiecki, H. Jakubowski
Analysis of site-specific N-homocysteinylation of human serum albumin 6 in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry.
Journal of Proteomics 74, 967-974 (2011).
P. Bednarek, M. Piślewska-Bednarek, E. Ver Loren van Themaat1, R.K. Maddula, A. Svatos, P. Schulze-Lefert
Conservation and clade-specific diversification of pathogen-inducible tryptophan and indole glucosinolate metabolism in Arabidopsis thaliana relatives.
New Phytologist 192, 713-726 (2011).
M. Delgado-Cerezo, C. Sanchez-Rodr?guez, V. Escudero, E. Miedes, P.V. Fernandez, L. Jorda, C. Hernandez-Blanco, A. Sanchez-Vallet, P. Bednarek, P. Schulze-Lefert, S. Somerville, J.M. Estevez, S. Persson, A. Molin
Arabidopsis Heterotrimeric G-protein Regulates Cell Wall Defense and Resistance to Necrotrophic Fungi.
Molecular Plant 5, 98-114 (2012).
Chemical warfare or modulators of defence responses - the function of secondary metabolites in plant immunity.
Current Opinion in Plant Biology 15, DOI 10.1016/j.pbi.2012.03.002, (2012)