Instrumental Methods in Biochemistry   (434B2)

Academic year:

2024/2025.

Attendance requirements:

302B2 + 401B2 + 221B2

ECTS:

7

Study level:

Study program:

Teacher:

Tanja D. Ćirković Veličković, Ph.D.
full professor, Faculty of Chemistry, Studentski trg 12-16, Beograd

Assistants:

—

Hours of instruction:

Weekly: two hours of lectures + four hours of labwork (2+0+4)

Goals:

This course is intended for the students who want to acquire knowledge and get practical training in working with the instruments used in biochemistry to determine the structure and modifications of proteins and in the study of ligand-macromolecule interactions (proteins and DNA).

Outcome:

Students are able to apply modern instrumental methods which are used to determine the structure of proteins and study ligand-macromolecule interactions.

Teaching methods:

Lectures, laboratory classes, progress tests, term papers.

Extracurricular activities:

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Coursebooks:

• J. R. Lakowicz: Principles of Fluorescence Spectroscopy, Springer 2006.
• J. Throck Watson, O. D. Sparkman: Introduction to Mass Spectrometry, Wiley 2007.
• G. D. Fasman: Circular Dichroism and the Conformational Analysis of Biomolecules, Springer, 1996.
• Nina Berova, Koji Nakanishi, Robert W. Woody: Circular Dichroism: Principles and Applications, Wiley, 2000.
• J. Lovric: Introducing Proteomics: From Concepts to Sample Separation, Mass Spectrometry and Data Analysis, Wiley, 2011.
• Lecture notes and lab notes.

Additional material:

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Lectures:

10 points (2 hours a week)

Syllabus:

1. An overview of modern instrumental methods in biochemistry: spectrofluorimetry, CD spectroscopy, microcalorimetry, FTIR, SPR, mass spectrometry.
2. Proteomic and metabolomic techniques.
3. The application of fluorescence spectroscopy in biochemistry. Natural fluorophores in proteins. The effect of the protein environment on the tryptophan emission. The application of spectrofluorimetry in the determination of protein-ligand interactions. Tryptophan fluorescence quenching method. The tertiary structure and fluorescence of proteins.
4. - The definition of circular dichroism in biochemistry, the concept of the secondary and tertiary structures of proteins; determination of the conformation of proteins and nucleic acids; determination of the conformational changes during protein-protein, protein-ligand, protein-DNA, DNA-ligand interactions.
   - Determination of binding constants by using the following methods: direct titration, serial dilution of complexes, the stability change during chemical and thermal denaturation.
5. The analysis of CD spectra by: the Spectrum Manager software package, the determination of protein secondary structure by using the CDPro software package.
6. - Mass spectrometry methods in proteomics.
   - Methods which can be coupled with mass spectrometry in proteomics: nanoscale liquid chromatography (HPLC), capillary electrophoresis, two-dimensional electrophoresis.
   - Sample preparation for mass spectrometry. "Bottom up proteomics": identification and characterization of proteins by "peptide mapping", de novo sequencing, the analysis of post-translational modifications (hydroxylation, phosphorylation), disulfide mapping, protein quantification (by the SILAC, iTRAQ, label-free quantification methods), the analysis of multiprotein complexes and cell organelles. Immunoproteomics. "Top-down proteomics": determination of the exact mass of proteins and peptides, the analysis of post-translational modifications (glycosylation, phosphorylation).
   - The analysis of mass spectra using software packages and algorithms: Mascot, Sequest, Profound, X!tandem, MassMatrix, ProSight.
   - The application of mass spectrometry in protein quantification of complex samples and biological fluids.
7. Mass spectrometry methods in metabolomics. The analysis of small molecules. Metabolic modifications of small molecules. The application of metabolomics in pharmacodynamic studies.

Labwork:

20 points (4 hours a week)

Syllabus:

1. Recording fluorescence spectra of proteins.
2. Determination of the binding constant for retinol bound to beta-lactoglobulin.
3. Determination of the binding constants for the selected polyphenols bound to beta-lactoglobulin by using the tryptophan fluorescence quenching method.
4. Determination of the melting point of proteins by using the spectrofluorimetric method: preparation and characterization of I, S and N forms of ovalbumin by spectrofluorimetry and CD spectroscopy.
5. Recording CD spectra of proteins and determining the secondary-structure contents in proteins.
6. Tracking the conformational changes of proteins during thermal denaturation by CD spectroscopy.
7. Tracking the conformational changes of proteins during denaturation by a chaotropic agent by using CD spectroscopy.
8. Determination of the exact mass of proteins by using mass spectrometry.
9. The peptide mapping, the peptide mass fingerprinting (PMF) of proteins and proteomics
   - reduction and alkylation of the samples in a solution;
   - sample preparation for PMF after 2D electrophoresis;
   - endoproteinase digestion;
   - chromatographic separation of peptides using nanoscale reversed-phase chromatography and the analysis of peptides using the LTQ Orbitrap XL mass spectrometer;
   - the analysis of mass spectra by using the following software packages and algorithms: Mascot, Sequest, X!tandem, Profound, MassMatrix, ProSight.
10. Determination of post-translational modifications of proteins.
    - Hydroxylation, phosphorylation.
    - Determination of the position of disulfide bridges in proteins.
11. Quantitative proteomics – determination of protein concentration in a mixture.
    - Relative and absolute quantification.
    - Using Skyline software.

Colloquia:

20 points

Written exam:

50 points