Research

Research AreaMolecular Biometry

Molecular Biometry is the application to biological research of quantitative analysis performed at the molecular level. Both proteomics and metabolomics are within the scope of Molecular Biometry.

Proteomics

Proteomics is large-scale analysis of the levels of expressed proteins and/or the occupancy their modifications. Our goal is to perfect the proteomics analysis and expand its application to a broad range of biomedical problems. We envision time when proteomics analyses will be widely used in clinical practice.  In order to achieve that, we work on solving a number of problems, such as:

  • Reliable and reproducible sample preparation, which requires robotization of sample handling and digestion. We have two robots for sample preparation.
  • Protein separation. We develop a novel tool for in-solution, on-line separation of proteins and peptides by their isolelectric point (pI).
  • Informative mass spectrometric analysis. We are primarily using high-resolution, high-mass accuracy measurements by Fourier Transform instrumentation (Orbitraps). In order to increase the informative content of tandem mass spectra, we develop novel fragmentation techniques, which are based on electron-ion interactions.
  • Accurate quantification. We develop the program Quanti that, uniqouely, can compensate in silico the fluctuations in the electrospray current and variations in instrumentalsensitivity during the LC/MS runs, ensuring high accuracy of label-free quantification.
  • Statistical analysis. We widely use correlation and principal component analysis, and further develop predictive bioinformatic tools.

Metabolomics

In this new for us area, we strive to provide complementary to proteomics information on cellular processes.

Pathway Analysis

Pathway analysis is a brainchild of Systems Biology. It integrates the signal coded in the abundances of hundreds of proteins to provide the activation levels of signaling as well as metabolic pathways, and to reveal the most important controlling molecules (key nodes) which can be used as drug targets. Together with our friends and collaborators Alexander Kel (GeneXplain) and Pedro Fernandes (Gulbenkian Institute of Biology), we have established in 2008 a permanent forum for wider application of pathway analysis in proteomics (PathProt [1]). The goal of these efforts is to develop the pathway search engine [2] – a bioinformatic tool that performs reliable, quantitative pathway analysis from a variety of –omics inputs.

Research Area Mass Spectrometry

Mass spectrometry is our main approach to studying nature. We believe in high resolution and high accuracy in mass and abundance measurements. In MS/MS, it appears that one fragmentation technique, regardless its mechanism, is insufficient for informative analysis, e.g. de novo sequencing [3]. The following aspects we deem particular important and work on them:

  • Continuing the multi-year effort in electron capture dissociation (ECD) and related techniques, develop novel fragmentation techniques that are based on charge increase as well as charge neutralization.
  • Studying the mechanism of ECD.
  • Studying “silent” (zero-mass) modifications [4].
  • Increasing the speed of analysis through MS/MS spectra multiplexing [5].
  • Improving the accuracy of label-free abundance measurements.

Research Area Isotopic Resonance Hypothesis

Isotopic Resonance Hypothesis [6] suggests that at certain “resonance” abundances of stable isotopes, the rate of chemical reactions of certain classes of compounds accelerates through the reduction of complexity of the system. Abiotic production of complex molecules, amino acids and peptides, from simple compounds could have been assisted by the presence in the inner Solar system of such an “isotopic resonance”. To test this hypothesis [7], we investigate whether rates of chemical and biochemical reactions depend non-linearly upon the isotopic compositions. We perform:

  • Miller experiment at different isotopic conditions.
  • Growth of microorganisms in different isotopic environment using highly accurate growth curve measurements.

Research Area Protein Separation

While reversed-phase chromatography of peptides and proteins is well integrated in the workflow of proteomics, there is a need for an orthogonal method of in-solution, on-line, separation by isolelectric point (pI). We develop such a method that affords separation of relatively large amounts of protein.

Research Area Fundamental Biology

We are interested in a variety of fundamental problems in Biology, particularly those related to two issues: a) Complexity, and b) Reversibility. There is a list of problems we deem particularly important; many of them are tightly inter-connected.

Research Area Deprivation Biology

How does deprivation of cells and bacteria of a certain element or amino acid affect their proteome, metabolome, as well as signaling and metabolic pathways? Can these effects be used for cancer treatment (e.g. arginine deprivation)?

Research Area Medicine

Reumatoid Arthritis (RA): as part of the SFF PRIMI consortium, we investigate the hypothesis that citrullination (deimination of arginine) is the protein modification central to RA mechanism.  We develop methods of sensitive and specific identification and quantification of citrullination in biopsies. We also quantify the glycosylation pattern of RA-related antibodies.

Alzheimer’s Disease (AD): as part of the FP7 PredictAD consortium, we investigated the blood plasma proteome of 218 age-matched individuals from the Kuopio cohort and found AD-predicting patterns in the abundances of 120-130 most abundant blood proteins. We also linked the amount of damaged proteins (aspartate isomerized to isoaspartate) in blood plasma with dementia [8]. In collaboration with the group of Prof. Thomas Bayer from the Göttingen University, we perform proteomics analysis of brains of AD mouse models, and have found molecular signatures of degradation before the onset of symptoms in young mice.

Cancer: we analyze the proteomes of six breast cancer cell lines (five of them – triple negative, basal type) to reveal common features as well as differences at the proteome level as well as at the level of signaling pathways. We also collaborate with Prof. Neus Visa of Stockholm University on the mechanism of 5-fu action on colon cancer cells.

Inflammation: in collaboration with Amgen and Bob Harris at KI, we study the response of monocytes on different pro- and anti-inflammatory stimuli with the aim to establish the proteomics-based inflammation scale.

References and Links

1. http://gtpb.igc.gulbenkian.pt/PathProt/

2. Marin-Vicente, C.; Zubarev, R. A. Search engine for proteomics, Fact or Fiction? G.I.T. Lab J, 2009, 11-12, 10-11.

3. Zubarev, R. A.; Zubarev, A. R.; Savitski, M. M. Electron Capture/Transfer versus Collisionally Activated/Induced Dissociations: Solo or Duet? J. Am. Soc. Mass Spectrom. 2008, 19, 753-761.

4. Yang, H.; Fung, Y. M.; Zubarev, A. R.; Zubarev, R. A.  Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples. J. Proteome Res. 2009, 8, 4615–4621.

5. Ledvina A. R.; Savitski M. M.; Zubarev A. R.; Good, D. M.; Coon, J. J.; Zubarev, R. A. Increased Throughput of Proteomics Analysis by Multiplexing High-Resolution Tandem Mass Spectra, Anal. Chem. 2011, 83, 7651-7656.

6. Zubarev, R. A.; Artemenko, K. A.; Zubarev, A. R.; Mayrhofer, C.; Yang, H.; Fung, E. Y. M. Early life relict feature in peptide mass distribution, Cent. Eur. J. Biol. 2010, 5, 190-196.

7. Zubarev, R. A. Role of Stable Isotopes in Life -  Testing Isotopic Resonance Hypothesis, Genomics, Proteomics & Bioinformatics, 2011, 9, 15-20.

8. Yang, H.; Lyutvinskiy, Y.; Soininen, H.; Zubarev, R. A. Alzheimer’s disease and mild cognitive impairment are associated with elevated levels of isoaspartyl residues in blood plasma proteins, J. Alzheimer’s Disease, 2011, 27, 113-118.