CG090 Methods in proteomics

Faculty of Science
Spring 2021
Extent and Intensity
2/0/0. 2 credit(s) (plus 2 credits for an exam). Type of Completion: zk (examination).
Teacher(s)
Mgr. Radka Dopitová, Ph.D. (lecturer)
doc. Mgr. Jan Havliš, Dr. (lecturer)
doc. Mgr. Ctirad Hofr, Ph.D. (lecturer)
Mgr. Tomáš Klumpler, Ph.D. (lecturer)
RNDr. Hana Konečná (lecturer)
doc. Mgr. Karel Kubíček, PhD. (lecturer)
Mgr. Gabriela Lochmanová, Ph.D. (lecturer)
doc. Mgr. Jan Paleček, Dr. rer. nat. (lecturer)
Mgr. David Potěšil, Ph.D. (lecturer)
prof. RNDr. Zbyněk Zdráhal, Dr. (lecturer)
Guaranteed by
prof. RNDr. Zbyněk Zdráhal, Dr.
National Centre for Biomolecular Research – Faculty of Science
Contact Person: prof. RNDr. Zbyněk Zdráhal, Dr.
Supplier department: National Centre for Biomolecular Research – Faculty of Science
Timetable
Mon 1. 3. to Fri 14. 5. Mon 15:00–16:50 C02/211
Prerequisites
basic knowledge of analytical chemistry, biochemistry and molecular biology
Course Enrolment Limitations
The course is also offered to the students of the fields other than those the course is directly associated with.
fields of study / plans the course is directly associated with
there are 6 fields of study the course is directly associated with, display
Course objectives
major goal of this subject is to acquaint students overall and generally with methods used in proteomics.
Learning outcomes
at the end of the course students should be able:
to explain principles of basic methods of proteomic analysis
to propose appropriate methods for analysis of given sample type with respect to purpose of analysis.
Syllabus
  • lesson 1 Jan Havliš
  • Basics in fractionation/separation of proteins and peptides – separation principles, physical-chemical properties of peptides and proteins, basic methods of protein isolation from different sample types; electrophoretic separation techniques (IEF, SDS-PAGE, 2-D gel electrophoresis, DIGE); liquid chromatography techniques (IEC, RPLC, HILIC, HIC, SEC), prediction of separation properties
  • lesson 2 Jan Havliš
  • Affinity chromatography (IMAC, MOAC, lectin LC), non-column separation techniques; multi-dimensional approaches for analysis of complex protein samples (2D methods and hyphenated techniques); protein sample preparation (input, basic CIPP approach, centrifugation, procedure control), example of enzyme preparation, example of S. cerevisiae proteome preparation, protein equalisers, commercial isolation kits
  • lesson 3 Gabriela Lochmanová, Hana Konečná
  • 2-D gel electrophoresis and image analysis in detail – preliminary course for practical exercises
  • lesson 4 Radka Dopitová
  • Introduction – recombinant proteins, general approach to their preparation, criteria for a selection of the expression system+ expression systems – bacterial, yeast, systems using insect cells and baculovirus vectors, others
  • lesson 5 Radka Dopitová
  • Basic methods of biomass disintegration; purification of recombinant proteins – basic methods for protein isolation (affinity, ion exchange, hydrophobic and gel permeation chromatography), principles for sequencing of methods; affinity anchors and purification, basic anchor removal procedures
  • lesson 6 Tomáš Klumpler
  • Methods of macromolecular structural analysis – crystallography, basic approaches to protein crystallography and small-angle X-ray scattering – macromolecular crystallization techniques, diffraction experiment, the phase problem & methods of its solving, electron density maps, structural model & its refinement
  • lesson 7 Karel Kubíček
  • Basics of NMR - short introduction, recapitulation of basic principles of nuclear spin, magnetic field, hardware, continuous wave vs. Fourier Transform NMR; NMR in studies of structure and dynamics of biomolecules; Studies of interaction using NMR; Intrinsically disordered proteins studied with 13C detected NMR
  • lesson 8 Jan Paleček
  • Protein-protein interactions analysis: matrix/beads using methods (pull-down, co-immunoprecipitation), hybride systems (classical, completion, membrane), proximity-based methods (FRET, PLA), protein-protein interactions mapping (reverse Y2H, peptide libraries, protein painting, H-exchange)
  • lesson 9 Ctirad Hofr
  • Binding curve; equilibrium dissociation constant; detector linear range; determination of binding affinity of fluorescently labelled proteins - fluorescence anisotropy, microscale thermophoresis; detection of binding of surface immobilized molecules - surface plasmon resonance, ELISA; study of binding of unmodified proteins directly in solution - isothermal titration calorimetry; case studies of quantitative methods use for protein-protein interaction analysis; summary of practical advantages and disadvantages of individual methods.
  • lesson 10 Zbyněk Zdráhal
  • Mass spectrometry of proteins - basic types of ionization techniques (ESI and MALDI) and MS instrumentation (ion trap, TOF, Orbitrap, hybrid instruments)
  • lesson 11 Zbyněk Zdráhal
  • protein identification methods (bottom-up, top-down); characterization of protein modifications (e.g. phosphorylations, acetylations, ubiquitinations); methods of protein quantification (relative and absolute quantification techniques, with utilization of tags, label-free approach)
  • lesson 12 David Potěšil
  • MS/MS data in proteomics (bottom-up vs. top-down proteomics, examples of proteomic bottom-up experiments), preparation of raw MS/MS data, selection and preparation of protein database, large-scale MS/MS data database searching, database search results processing, list of proteins present in the sample reconstruction, protein quantification and analysis of quantitative information, proteins lists interpretation (biological networks).
Literature
    recommended literature
  • BUDINSKA, Eva, Otakar FOJT and Ladislav DUŠEK. Bioinformatics in Genomics and Proteomics Data. 2009. URL info
  • TWYMAN, R.M. Principles of proteomics. BIOS Scientific Publishers, 2008. ISBN 1 85996 273 4. info
  • EIDHAMMER, Ingvar. Computational methods for mass spectrometry proteomics. Chichester: John Wiley & Sons, 2007, x, 284. ISBN 9780470512975. info
  • CAMPBELL, A. Malcolm and Laurie J. HEYER. Discovering genomics, proteomics, and bioinformatics. 2. ed. San Francisco: CSHL Press, 2007, xv, 447. ISBN 0805382194. info
  • Mass spectrometry data analysis in proteomics. Edited by Rune Matthiesen. Totowa, N. J.: Humana Press, 2007, x, 320. ISBN 9781588295637. info
  • THIELLEMENT, Hervé. Plant proteomics : methods and protocols. Totowa, N.J.: Humana Press, 2007, xiii, 399. ISBN 9781597452274. info
  • SIMPSON, Richard J. Proteins and proteomics : a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2003, xiii, 926. ISBN 0879695544. info
  • Posttranslational modifications of proteins : tools for functional proteomics. Edited by Christoph Kannicht. Totowa, N.J.: Humana Press, 2002, xi, 322. ISBN 0896036782. info
  • Proteomics. Edited by Timothy Palzkill. Boston: Kluwer Academic Publishers, 2002, viii, 136. ISBN 0792375653. info
Teaching methods
The lecture is based on ppt presentations and their explication. Presentations themselves will be available as study materials (black-and-white printable pdf with high resolution and restricted access rights). It is recommended to attend the lecture, because of the explication, which significantly extends the presentation and because there are no available textbooks in Czech language covering certain parts of the subject. Students will test their acquired knowledge by means of "in-rough" tests, which will have similar form and content as the final examination test, they will be corrected to give the feed-back, but not graded.
Assessment methods
written examination; test consists of sixty questions with five choices per question with always only one correct answer; to pass the test, it is necessary to answer correctly at least 60 % of the questions; test covers complete content of the course
Language of instruction
Czech
Further comments (probably available only in Czech)
Study Materials
The course is taught annually.
Listed among pre-requisites of other courses
Teacher's information
videocon lectures will take place in the MS teams environment. if possible, download the environment application. all MU students have access to it via office 365, see "external services" in IS. i recommend learning to work with the application in advance. the use of a browser version restricts your access to some useful functions. the lecture will be recorded in mp4 format and then uploaded in the study materials in IS. MS teams guides: https://is.muni.cz/auth/do/mu/samostudium/pages_en/02-online.html
The course is also listed under the following terms Spring 2020, Spring 2022, Spring 2023, Spring 2024, Spring 2025.
  • Enrolment Statistics (Spring 2021, recent)
  • Permalink: https://is.muni.cz/course/sci/spring2021/CG090