Laboratory of mass spectrometry

                                                            


 

Head:
Dr. Łukasz Marczak

 

 


Staff:
 Dr. Marta Sikora,

Jakub Idkowiak

 

 


PhD Students:
Izabela Bielińska, MSc;

Joanna Tracz, MSc;

Urszula Strybel, MSc

 

 


 

Rys.1

 


Rys.2


Rys.3


Rys.4

 


 

 

 

 

Research offer:

- protein identification using MS methods

- peptide or protein sequence de novo analysis (MALDI, ISD-MALDI)

- analysis of protein posttranslational modifications using MS methods

- quantitative analysis of proteins using electrophoretic and MS methods

- HR-MS analysis of low molecular mass compounds (exact monoisotopic mass measurement)

- structural elucidation of compounds using tandem mass spectrometry

- quantitative analysis of compounds using MS techniques

- analysis of volatile compounds using GC-MSn

 

Equipment:

Institute of Bioorganic Chemistry

-              HPLC-MS system (Q-TOF) - Bruker micrOTOF-q

-              MALDI-TOF mass spectrometer - Bruker Autoflex

-              GC-MS (TOF) system - Waters GCT Premier

European Centre for Bioinformatics and Genomics:

-              nanoLC-MS system (offline MALDI-TOF/TOF) – Proxeon nanoLC + Bruker UltrafleXtreme

-              nanoLC-MS system (ion trap) – Waters nanoAcquity + Bruker Amazon SL

-              nano/micro LC-MS system (OrbiTrap) – Dionex RSLC nano 3000 + Thermo QExactive

-              GC x GC –MS system (TOF)– Leco Pegasus 4D

-              GC-MS system (TripleQuad) – Thermo TSQ8000

-              standalone nano ion source with fraction collector - Advion NanoMate TriVersa

-              2D gel electrophoresis system - GE Healthcare IPGphor + EttanDalt six

 

Scientific profile:

Application of mass spectrometry techniques for qualitative and quantitative proteomic analysis. We use techniques based on isotopic/isobaric labelling of peptides of interest as well as label free techniques for quantitation. Due to combination of modern LC-MS/MS with broad range of dedicated advanced software we are able to precisely identify thousands of proteins within one sample.

Qualitative and quantitative analyses of low molecular weight compounds are performed based on modern separation techniques (liquid chromatography, 2D gas chromatography) combined with advanced detectors for identification of molecules of interest (MS). Ongoing research relies mainly on separation of complex mixtures of natural compounds together with their detection and standardization leading to information about sample composition and possibility to differentiate particular compounds between samples.

Current research activity

Current ongoing projects are focused mainly on analysis of blood proteome in different stages of human diseases. We are dealing with clarifying the molecular basis of the intensification of atherosclerotic lesions in patients with chronic kidney disease. We want to understand the factors underlying the observed differences between the proteomic profiles of patients suffering from classical atherosclerosis and arteriosclerosis co-occurring with kidney damage. We analyze the impact of disease progression as well as various regimens on the observed proteome.

An important object of study are also human cancers such as acute myeloid leukemia, multiple myeloma and larynx cancer. We analyze plasma, serum and blood cell fractions (granulocytes, plasma cells). Tests are performed using the cells cultured in vitro, derived from primary tumors and metastases.

Until now, the mechanism underlying the pathophysiology of hyperhomocysteinemia (HHcy) in cardiovascular diseases or brain is unknown. The aim of the research conducted in our laboratory is to clarify the mechanisms of the process of N-homocysteinylation of histone proteins in vivo in the context of deficiency of cystathionine beta-synthase (CBS) and the relationship of this process with the mechanisms underlying the toxicity of Hcy. The main hypothesis that N-homocysteinylation of histones is enhanced under conditions leading to cardiovascular disease, ie. HHcy, and is responsible for the pathophysiology, is verified through the following specific objectives:

1) Analysis of N-homocysteinylation process of histones in vivo, in tissue from different organs taken from the mouse with a mutation in the Cbs gene

2) Identification of lysine residues of histone undergoing specific N-homocysteinylation in vivo

3) Analysis of the proteome of organs form Cbs -/- and Cbs +/+ mice

4) Analysis of the impact of histones N-homocysteinylation on their interaction with DNA

A mouse model of protein N-homocysteinylation is also used by us to investigate systemic changes at the level of the proteome and the coagulation cascade. We also examine quantitative changes of mouse plasma proteins N-homocysteinylation to observe the correlation between serum total homocysteine and the degree of modification of these proteins.

Analysis of plant phenolic compounds for the identification of new derivatives. The use of liquid chromatography and CID fragmentation to distinguish and identifiy flavonoid glycosides.

 

Most important research achievements:

  • development of new methods for qualitative and quantitative determination of proteins N-homocysteinylation,
  • identification of marker proteins of hyperhomocysteinemia in the kidneys of mice with knockout of gene encoding CBS,
  • studies on changes in peptide and protein profiles of samples of human blood in cancer patients,
  • identification of marker proteins in blood samples from patients with chronic kidney disease
  • identification of new derivatives of flavonoids in plant extracts

 

Actual research projects:

•     Analysis of blood plasma proteome in patients with chronic kidney disease in the context of the progression of cardiovascular disease. Implementation period: 2013.03.01 – 2016.09.30

•     The search for molecular mechanisms of progression of atherosclerosis in patients with chronic kidney disease. Implementation period: 2016.06.20 – 2019.06.19

•     The pathophysiological consequences of histones N-homocysteinylation in hyperhomocysteinemia caused by deficiency of CBS. Implementation period: 25.08.2015 - 24.08.2016

•     Survival and oxidative stress in hyperhomocysteinemia. Implementation period: 2016.01.28 – 2019.01.27

 

Selected publications:

M. Sikora, L. Marczak, T. Twardowski, M. Stobiecki, H. Jakubowski, (2010) Direct monitoring of albumin lysine-525 N-homocysteinylation in human serum by liquid chromatography/mass spectrometry. Analytical Biochemistry 405, 132-134.

L. Marczak, M. Sikora, M. Stobiecki, H. Jakubowski (2011) Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry. Journal of Proteomics 74, 967-974

M. Łuczak, D. Formanowicz, E. Pawliczak, M. Wanic-Kossowska, A. Wykretowicz, M. Figlerowicz (2011) Chronic kidney disease-related atherosclerosis - proteomic studies of blood plasma. Proteome Science, vol. 9: art. no. 25.

M. Luczak, M. Kaźmierczak, L. Handschuh, K. Lewandowski, M. Komarnicki, M. Figlerowicz (2012) Comparative proteome analysis of acute myeloid leukemia with and without  maturation. Journal of Proteomics 5734-5748.

M. Kaźmierczak, M. Luczak, K. Lewandowski, L. Handschuh, A. Czyż, M. Jarmuż, M. Gniot, M. Michalak, M. Figlerowicz, M. Komarnicki (2013) Esterase D and gamma 1 actin level might predict results of induction therapy in patients with acute myeloid leukemia without and with maturation. Medical Oncology 30:725.

M. Luczak, Ł. Marczak, M. Stobiecki (2014)  Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. PLOS One. 9; e101694

M. Sikora, L. Marczak, J. Kubalska, A. Graban, H. Jakubowski (2014) Identification of N-homocysteinylation sites in plasma proteins. Amino Acids 46, 235-244

M. Luczak, Ł. Marczak, D. Formanowicz, E. Pawliczak, M. Wanic-Kossowska, A. Wykretowicz, M. Figlerowicz, M. Stobiecki (2015) Deeper insight into chronic kidney disease-related atherosclerosis: comparative proteomic studies of blood plasma using 2DE and mass spectrometry. Journal of Translational Medicine. 13:20

Marczak Ł., Znajdek-Awiżeń P., Bylka W (2016) The Use of Mass Spectrometric Techniques to Differentiate Isobaric and Isomeric Flavonoid Conjugates from Axyris amaranthoides. Molecules, 21, Art. No.1229.

Łuczak M, Formanowicz D, Marczak Ł, Suszyńska-Zajczyk J, Pawliczak E, Wanic-Kossowska M, Stobiecki M (2016) iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis. Scientific Reports, 6, Art. No. 32 511.

Kachlicki P, Piasecka A, Stobiecki M, Marczak Ł (2016) Structural characterization of flavonoid glycoconjugates and their derivatives with mass spectrometric techniques. Molecules, 21, Art. No. 1494

Luczak, M., Suszynska-Zajczyk, J., Marczak, L., Formanowicz, D., Pawliczak, E., Wanic-Kossowska, M., Stobiecki, M (2016) Label-Free Quantitative Proteomics Reveals Differences in Molecular Mechanism of Atherosclerosis Related and Non-Related to Chronic Kidney Disease. International Journal of Molecular Sciences, 17, Art No. 631