Content of lectures: 1. Basics of mass spectrometry (basic terminology, units, physical constants). The principles of the substances ionization and dissociation of ions (ionization in gas phase by energetic electrons), formation of ions, stable, metastable, and unstable ions. 2. Practical aspects of electron ionization. Isotopic composition and exact molecular weight of the substances (mono -isotopic, di- and poly- isotopic elements, calculating the distribution of isotopes). Exact molecular weight and composition of molecules (mass defect, accuracy and reliability of data). Application of high resolution mass spectrometry. 3. Instrumentation (quadrupole, linear and 3D ion trap, magnetic sector instruments, time of flight analyzers, FT ion cyclotron resonance, Orbitrap, detectors). 4. Other methods of ionization (chemical ionization, field ionization) tandem mass spectrometry, FAB, MALDI, ambient MS (ESI, APCI, APPI) DESI DART. Metabolomics based on mass spectrometry, metabolomic database. 5. Magnetic moments of nuclei, nuclei used in NMR spectroscopy of proteins and nucleic acids, isotopic labeling. NMR spectrometer, homogenous static magnetic field, radio frequency pulses, the resonant frequency, excitation, magnetization, polarization, coherence. Interaction of magnetic moments with molecular surroundings, dipole-dipole interactions with the surrounding nuclei and nuclear Overhauser effect (NOE), shielding the electrons and the chemical shift, the interaction with the surrounding nuclei mediated by bonding electrons (scalar interactions). Basic NMR experiment, Fourier transformation. 6. Strategy for proteins and nucleic acids study by NMR. Multivariate spectroscopy, heteronuclear and homonuclear correlation, 3D experiments for assigning resonant frequencies for proteins cores, assignment side chains, assignment of frequencies to the centre of nucleic acids. 7. NMR experiments providing information on the structure, distance of atoms from NOE, experiment NOESY, torsion angles from three bonding scalar coupling constants, orientation of molecular fragments from the residual dipole interaction, partially oriented samples, determination of secondary structure, calculation structural models from NMR data. 8. Dynamics of proteins and nucleic acids, the NMR relaxation, correlation functions, spectral density. Rotational diffusion, rapid internal motions, order parameter, correlation times, conformational and chemical exchange, experiments for the description of molecular motions and interactions between molecules. 9. Physical principles of electron microscopy, the physical properties of accelerated electrons, resolution, magnification, transmission electron microscope. 10. Preparation of specimens for TEM by physical and chemical methods. 11. Scanning electron microscopy and preparation of specimens for SEM. 12. Cytology using electron microscopy. Content of practicals: 1. Analysis of the basic NMR experiment, the evaluation of the simulated NMR spectrum, Fourier transformation. 2. Frequency assignment to peptide backbone based on the simulated 3D spectra, sequential assignment of DNA fragment based on simulated NOESY spectra. 3. Determination of protein secondary structure based on NMR data. Evaluation of relaxation data, determination of the parameters describing the movements of molecules.
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Jürgen H. Gross: Mass Spectrometry. A textbook, Second Edition, Springer Verlag Berlin, Heidelberg 2011.
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Selvin P.R., Ha T.: Single-molecule techniques, CSH Press, New York, 2008.
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