1. Proteome

2. Proteome Set of all protein in certain system
GENOME
Transcriptome
Nucleus
Nuclear membrae
Proteome
Proteome
Mitochondria ER
Golgi
Membrane
Vesicles
Chloroplasts
Vacuole
Set of all protein in certain system
- organelles, cells, tissues, organs, organisms, treatments, etc.
- includes all protein states (e.g. posttranslational modifications = protein encoded by a single gene is present in many functionally different forms)
- e.g. Homo sapiens genes, but estimated approx proteins (protein forms)

3. Analysis of protein level and localization
Detection of presence of certain protein
- by activity determination (possible for some enzymes)
- immunodetection: Western blot (after SDS-PAGE), ELISA, …
Analysis of certain protein localization
- translational fusion with reporter gene (GFP); in situ immunodetection
Proteome analysis/comparisons
2D-electrophoresis
„Gel-free“ methods

4. Detection of selected protein on Western blot
Protein electrophoresis (SDS-PAGE)
transfer of proteins from gel onto membrane = Western blot
Detection by antibodies
(primary = interacts with the protein
secondary = interacts with primary antibodies from certain species
vizualization usually by enzymatic reaction (color precipitate, chemiluminiscence)
W. blot
Secondary antibody conjugated with
an enzyme or fluorescent label
Primary
antibody
Membrane with bound proteins

5. Translational fusion with GFP
Removal of stop codone and attachement of GFP gene in reading frame
(GFP without stop codone can be even in N-terminus if there is not a localization signal)

6. Translational fusion with GFP
Studies of protein localization and protein interactions,
Monitoring of different processes and structures in living cells …
Golgi apparatus chromozomes microtubules

7. Physiological state of cell
Proteomics - factors influencing proteome
Cell cycle phase
Interactions with environment
Temperature
???
Proteome
Stress
Physiological state of cell
Cell specific gene expression
Genome

8. Proteome analysis
Before identification, proteins (peptides) has to be fractionated/separated – e.g. 2D-electrophoresis, …

9. Principle of 2-dimensional electrophoresis
IEF: isoelectric focusing
SDS-PAGE
Every spot represents one protein form (amino acid exchange or
postranslational modification can shift the position of protein on the gel)

10. First dimension: IEF (isoelectric focusing):
separation of proteins according their
isoelectric point (pH with zero charge)
proteins move to the position in gel, where pH
matches with their isoelectric point
separation of proteins by their charge in pH gradient

12. Second dimension SDS-PAGE
denaturation within the first dimension gel
separation of proteins by size in polyacrylamide gel

13. Principle of 2-dimensional electrophoresis
IEF: isoelectric focusing + -
pH 3
pH 10 -
200 kD
SDS-PAGE +
20 kD

14. Staining of protein gels
With silver (nitrate; sensitive, non- quantitative)
Coomassie blue (less sensitive, quantitative)
RI (in vivo labelïng)
Fluorescent dyes (DIGE)
proteome comparisons
difference in gel
electrophoresis

15. Protein identification (peptide fingerprinting)
Protein digestion with
trypsine or other protease (specific, reproducible cleavage!)
MS (e.g. MALDI/TOF)
Matrix-assisted
Laser desorption / ionisation
Time-of-flight analysis

16. MALDI Peptides are crystalized in matrix
Laser flash ionizates matrix molecules
Peptides are ionizated with protons transfered from matrix

17. MALDI ToF Matrix-assisted Laser desorption / ionisation Time-of-flight
Peptides are crystalized in matrix
Laser flash ionizates matrix molecules
Peptides are ionizated with protons
transfered from matrix
ToF
Time-of-flight
(reflects mass)

18. Protein identification by „peptide fingerprinting”
Protein digestion (by trypsine)
protein I (12 kD)
2 fragments (4 kD, 8 kD)
protein II (16 kD)
3 fragments (2 kD, 6 kD, 8 kD)
DNA (i EST), protein database
generation of theoretical
cleavage products
MALDI ToF
protein I
protein II
m/z
4000
8000
2000
6000
protein a: 3, 5, 9, 12 kD
protein b: 2, 7, 9 kD
protein c: 4, 8 kD
protein d: 3, 9, 12 kD
protein e: 2, 6, 8 kD
Comparison of experimental peptide sizes
with the theoretical set generated from proteins
in database

19. MS/MS peptide „sequencing“
by MALDI TOF/TOF
Izolated protein or protein mixture is cleaved with protease
(alt. LC separation of peptides from protein mixture)
MALDI TOF/TOF
Peptide selection (by TOF)
Peptide fragmentation (collision cell with lower vacuum)
TOF analysis of peptide fragments

20. Mass spectrum of peptide fragments

21. Preferential fragmentation in peptide bond
- splitting into two fragments

22. MS spectra analyses

23. (Isobaric tags for relative and absolute quantitation)
iTraq
(Isobaric tags for relative and absolute quantitation)
„Gel free“ method - an alternative approach to 2D electrophoresis allowing differential labelling and subsequent quantification of proteins from compared samples
- protein reproducible cleavage to peptides in each sample
- modification of peptides in each sample (chemically same group, but different nonradioactive isotypes)
- mixing samples, fractionation
- MS/MS analysis
- peptide identification
- determination of peptide quantity (ratios between samples)

24. iTraq label
Ross P L et al. Mol Cell Proteomics 2004;3:

25. - peptide identification (fragmentation spectrum) + - quantification (quantity/ratio of tags)

26. Example of model experiment
Modification of
amino groups with
iTraq reagents
4-(8) x
(every sample with
different reagent)
(every sample separately)
Mixing together
frakcionace směsi peptidů
MALDI TOF/TOF
(identification of peptides +
ratio of peptides originating
from different samples)
Isoelectric
focusing of peptides LC

27. Protein complex analysis
separation of native complexes (surfice-bound Coomassie BB)
by size in gradient gel
Blue Native Gel Electrophoresis
SDS-PAGE