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Proteome
GENOME
Transcriptome
Nucleus
Proteome
Nuclear
membrae
ER
ProteomeMitochondria
Golgi
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 30.000 genes, but estimated approx.
500.000 proteins (protein forms)
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
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
W. blot
vizualization usually by enzymatic
reaction (color precipitate,
chemiluminiscence)
Primary
antibody
Membrane with bound proteins
Secondary antibody conjugated with
an enzyme or fluorescent label
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)
Translational fusion with GFP
Studies of protein localization and protein interactions,
Monitoring of different processes and structures in living cells
…
Golgi apparatus
chromozomes
microtubules
Proteomics - factors influencing proteome
Cell cycle phase
Interactions with environment
Temperature
Proteome
???
Stress
Physiological state of cell
Cell specific gene expression
Genome
Proteome analysis
Before identification, proteins (peptides) has to be
fractionated/separated – e.g. 2D-electrophoresis, …
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)
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
Second dimension SDS-PAGE
- denaturation within the first dimension gel
- separation of proteins by size in polyacrylamide gel
Principle of 2-dimensional electrophoresis
IEF: isoelectric focusing
-
pH 3
pH 10
200 kD
SDS-PAGE
-
+
+
20 kD
Staining of protein gels
– With silver (nitrate; sensitive, nonquantitative)
– Coomassie blue (less sensitive,
quantitative)
– RI (in vivo labelïng)
– Fluorescent dyes (DIGE)
- proteome comparisons
- difference in gel
electrophoresis
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
MALDI
• Peptides are crystalized in matrix
• Laser flash ionizates matrix molecules
• Peptides are ionizated with protons
transfered from matrix
MALDI
Matrix-assisted
Laser desorption /
ionisation
• Peptides are crystalized in matrix
• Laser flash ionizates matrix molecules
• Peptides are ionizated with protons
transfered from matrix
ToF
Time-of-flight
(reflects mass)
Protein identification by „peptide fingerprinting”
Protein digestion (by trypsine)
protein I (12 kD)
DNA (i EST), protein database
2 fragments (4 kD, 8 kD)
protein II (16 kD)
3 fragments (2 kD, 6 kD, 8 kD)
generation of theoretical
cleavage products
MALDI ToF
protein I
4000
8000
m/z
protein a:
protein b:
protein c:
protein d:
protein e:
3, 5, 9, 12 kD
2, 7, 9 kD
4, 8 kD
3, 9, 12 kD
2, 6, 8 kD
protein II
2000
6000
8000
m/z
Comparison of experimental peptide sizes
with the theoretical set generated from proteins
in database
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
Mass spectrum of peptide fragments
Preferential fragmentation in peptide bond
- splitting into two fragments
MS spectra analyses
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)
iTraq label
Ross P L et al. Mol Cell Proteomics 2004;3:1154-1169
- peptide identification (fragmentation spectrum) +
- quantification (quantity/ratio of tags)
Example of model experiment
Modification of
amino groups with
iTraq reagents
4-(8) x
(every sample separately)
(every sample with
different reagent)
Mixing together
frakcionace směsi peptidů
MALDI TOF/TOF
(identification of peptides +
ratio of peptides originating
from different samples)
LC
Isoelectric
focusing of peptides
Protein complex analysis
- separation of native complexes (surfice-bound Coomassie BB)
- by size in gradient gel
Blue Native Gel Electrophoresis
SDS-PAGE