Course: Proteomics

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Course title Proteomics
Course code KBC/PROT
Organizational form of instruction Lecture
Level of course Master
Year of study not specified
Semester Summer
Number of ECTS credits 3
Language of instruction Czech, English
Status of course Compulsory, Compulsory-optional, Optional
Form of instruction Face-to-face
Work placements This is not an internship
Recommended optional programme components None
Lecturer(s)
  • Řehulka Pavel, RNDr. Ph.D.
  • Šebela Marek, prof. Mgr. Dr.
  • Lenobel René, Mgr. Ph.D.
Course content
Introduction to proteomics. Proteome. Basic terminology of protein separation and analysis. Branches of proteomics. Significance of expression, differential and functional proteomics. Partial separation of protein mixtures. Conventional chromatography, affinity chromatography, preparative electrophoresis. One-dimensional electrophoresis (1-D) - SDS/PAGE. Two-dimensional (2-D) electrophoresis. Sample preparation. First dimension - isoelectric focusing (IEF). Ampholytes, tube IEF gels. Gel strips with immobilized pH gradient ("IPG strips"). Methodology and instrumentation of IEF separation. Reversible staining of IPG strips. Second dimension - SDS/PAGE. Buffer composition, casting of gels. Instrumentation of SDS/PAGE (horizontal and vertical units). Protein staining in SDS/PAGE gels, compatibility of staining with mass spectrometry. Fluorescence staining of gels. Imaging, densitometry, software for evaluation of 2D gels. Comparison of two or more 2D gels, difference analysis. DIGE method. Excision of protein spots. Electroelution. Chemical and enzymatic digestion of proteins. In-gel digestion. Enzyme reactor, on-membrane digestion. General principles of mass spectrometry. Ionization techniques used in proteomics. Electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI). Mass analyzers, quadrupole (Q), ion trap (IT), time-of-flight analyzer (TOF), reflectron, Fourier transformed ion cyclotron resonance (FT-ICR). Tandem mass spectrometry (MS/MS). Hybrid instruments. Peptide mass fingerprinting (PMF), de novo sequencing of peptides. Sequence analysis using the method of post source decay (PSD). Protein identification by database searching. Searching tools for PMF and MS/MS. Organisms with unsequenced genomes, EST databases, MS Blast program. LC-MS/MS of peptides. Shotgun proteomics - a tool for analysis of complex protein mixtures. Multidimensional liquid chromatography (LC) and capillary electrophoresis (CE). LC-LC, LC-CE and CE-CE combinations. Multidimensional protein identification technology (MuD-PIT). Post-translational modifications (PTM). Affinity chromatography of phosphopeptides. Mass spectrometry of phosphopeptides and glycopeptides. Quantitative analysis in proteomics: H2O18, ICAT, SILAC and AQUA. Clinical proteomics. Biomarkers. Protein chips. SELDI MS. Sample purification on affinity materials. MALDI imaging. Characterization of microorganism by mass spectrometry.

Learning activities and teaching methods
Lecture
  • Attendace - 26 hours per semester
  • Preparation for the Exam - 50 hours per semester
Learning outcomes
Students will acquaint themselves with the history and orientation of proteomics, separation methods for proteins, mass spectrometry of proteins and peptides a with methods for identification and quantification of proteins.
To define main concepts, describe methods used in proteomics.
Prerequisites
It is necessary to have knowledge gained from successful completion of the courses KBC/BCH Fundamentals of Biochemistry, (KBC/UBCH Introduction to Biochemistry for students of Bioinformatics) and KBC/BIME Biochemical Methods.

Assessment methods and criteria
Written exam

Successful passing of written examination - obtaining of at least 60% of credit points. Evaluation: 20 - 18 p. A (1) 18 - 16.p B (1-2) 16 - 14 p. C (2) 14 - 12 p. D (2-3) 12 - 10 p. E (3)
Recommended literature
  • HILLENKAMP, F., PETER-KATALINIC, J. MALDI MS: A Practical Guide to Instrumentation, Methods and Application, Wiley-VCH, Weinheim, 2007..
  • TRAMONTANO, A. Protein Structure Prediction, Wiley-VCH, Weinheim, 2006..
  • WESTERMEIER, R. Electrophoresis in Practice, Wiley-VCH, Weinheim, 2001..
  • Westermeier, R., Naven, T. (2002). Proteomics in practice. A laboratory manual of proteome analysis.. Wiley-VCH, Weinheim, Germany.


Study plans that include the course
Faculty Study plan (Version) Branch of study Category Recommended year of study Recommended semester
Faculty of Science Biotechnology and Gene Engineering (1) Chemistry courses 1 Summer
Faculty of Science Bioinformatics (1) Informatics courses 1 Summer
Faculty of Science Biochemistry (1) Chemistry courses 1 Summer
Faculty of Science Molecular Biophysics (2015) Physics courses 1 Summer
Faculty of Science Bioinorganic Chemistry (1) Chemistry courses 1 Summer