Concepts of protein technology
Proteomics advantages:
Proteomics: determining the complete set of proteins that is present in a system under specific circumstances
System: protein complex, subcellular compartment, cell, tissue, organism
Circumstances: treatment, time after treatment, condition of the cell (age, tumour, infected)
System biology: proteomics as a part of system biology
mRNA + protein profiles and their change over time
Protein post-translationational modifications, cellular localization, ligand binding, alternative
splicing, degradation
Protein-protein interactions
Proteomics sensu strictu: large scale characterization of proteins (including posttranslationational
modification) = shotgun proteomics
Differential proteomics: large scale comparison of protein expression
Cell-mapping proteomics: protein-protein interaction studies
Traditional biochemistry: good to focus on 1 protein ↔ Proteomics: higher level of information
Functions that cannot get predicted by genome sequencing:
1. Genes don’t inform about organism complexity:
Genomes: number of genes is not giving enough information about the organism complexity
o Humans: 20.000 – 40.000 genes
o Yeast: 6.000 genes
o Drosophila: 13.000 genes
o Earth worm: 18.000 genes
2. No correlation between mRNA and protein profiling:
RNA sequencing: no direct correlation between mRNA amount and protein expression
sometimes high RNA amounts give low amounts of protein / low RNA gives much proteins
3. No direct protein-gene relation:
Proteins can be post-translationally modified
Alternative splicing: 1 gene can result in different isoforms bv α -1-antitrypsin has 22 isoforms
Proteoforms: different forms of the same protein arising from 1 SNP / differential splicing / post-
translational modification
Can be linked to diseases
4. Protein interaction networks:
Protein complexes: most cellular processes regulated by protein complexes
Functional proteomics: define protein as an element in an interaction network (don’t ascribe it 1 function)
5. Cellular localisation:
Cell localisation: proteins can be localised in nucleus, cytosol, plasma membrane, mitochondria, ER
1 protein can have different functions depending on their cellular localisation
,Introduction to proteomics protocol:
1. Protein extraction from sample
2. Protein separation
3. Protein digestion with protease
4. Peptide separation by liquid chromatography
5. Electrospray ionization brings peptides in
gas phase + gives positive charge
6. Ion mobility in mass spectrometry
7. Data analysis
Bottom-up proteomics: identify proteins based on the peptide mixture
(proteome -> proteins -> enzymatic digestion -> peptide mixture)
Top-down proteomics: immediately introduce purified proteins in mass spectrometer
(proteome -> MS analysis)
Sample preparation:
1. Break up tissues/cells and extract the protein fraction
2. Modify proteins based on further analysis steps (denaturation, reduction,…)
Variables for separation success:
Method of cell lysis, type of detergent
pH
Temperature
Proteolytic degradation
Protein digestion:
LC-MS done on peptides
Trypsin: hydrolyses the C-terminal of lysine and of arginine
Chymotrypsin: hydrolyses the C-terminal of large hydrophobic residues tyrosine, phenylalanine, tryptophane
Peptide separation:
Sample enrichment: some proteins reside in the cell in lower abundancy
11 orders of magnitude between the highest and lowest abundant protein
Lower abundance proteins cannot be seen in chromassy blue staining gel
Increase peak capacity:
Sequential separation techniques: each dimension made by a technique based on different
physicochemical properties of the proteins
Separation capacity: amount of peaks fitting into chromatogram without overlapping peaks
= maximum amount of components that can be separated by the chromatographer
Peak capacity: multiplication of separation capacities
Peak capacity=CS1∗CS 2∗CSn−1∗CS n
,Chromatography:
Chromatography: separates biomolecules on the basis of their distribution over the stationary and
mobile phase
Molecules with higher affinity for the stationary phase get slowed down
The longer chromatography runs the better the separation
Ion-exchange chromatography: stationary phase in neg/pos charged
Same charge molecules go through faster
Gel permeation / filtration chromatography: small molecules get stuck in the pores
Big molecules elute faster
Reverse phase chromatography: hydrophobic parts of the protein will bind to the hydrophobic stationary phase
Polar molecules elute faster
Ion pairing reagent: has hydrophobic tail and charged head to help more polar molecules
bind the stationary phase
Reverse phase used for proteomics
SDS PAGE
Mass spectrometry:
1. Ion formation: by ionization source that converts peptides in gaseous ions
2. Ion separation: separation based on the mass/charge ratio (m/z) by electric and magnetic
fields in the mass analyzer
3. Ion detection: specific current strength produced based on m/z ratio and abundancy of the ion
Data analysis:
De novo-sequencing: determining peptide AA sequence from the spectrum
In silico spectra: experimental MS spectra compared to in silico spectra generated from a protein database
Sample preparation:
, Intact proteins:
Gel based separation
Affinity purification by antibodies
Protein chips and arrays
Antibody based identification / LC-MS/MS
Purified intact proteins analysed by LC-MS after digestion gives most detailed info
Digested proteins:
Liquid chromatography
Mass spectrometry
Proteoform info can be lost after digestion
Protein interference problem: difficult to know which proteins/ proteoforms are in a complex once
it’s digested
→ Mass spectrometry: identifies protein groups
When identifying peptide 1 and 2 : the protein in the sample can be
A/B/C/D/E
Identify A if you know peptide 0 is in it
1. Define research question:
o Investigate protein, proteoform, protein class, proteome?
o Which techniques available?
o Tertiary structures / protein complexes intact?
2. Find best sample preparation protocol:
Protocol: tailored to extract a single protein , more specific research question results in more
optimal preparation protocol
Protein diversity: bv cytosolic proteins , membrane associated proteins , protein complexes , short
open reading frames , …
General steps:
1. Experimental design
2. Literature study
3. Sample collection
4. Protein extraction , solubilization
5. Protein separation or purification
6. Reduction and alkylation
7. Digestion
8. Peptide purification or selection
9. Prefractionation
Optimal sample type:
Lab grown cells not representative for real tissue
Patient samples more variable
Serum and plasma highly variable due to protein concentration differences
Proteomics advantages:
Proteomics: determining the complete set of proteins that is present in a system under specific circumstances
System: protein complex, subcellular compartment, cell, tissue, organism
Circumstances: treatment, time after treatment, condition of the cell (age, tumour, infected)
System biology: proteomics as a part of system biology
mRNA + protein profiles and their change over time
Protein post-translationational modifications, cellular localization, ligand binding, alternative
splicing, degradation
Protein-protein interactions
Proteomics sensu strictu: large scale characterization of proteins (including posttranslationational
modification) = shotgun proteomics
Differential proteomics: large scale comparison of protein expression
Cell-mapping proteomics: protein-protein interaction studies
Traditional biochemistry: good to focus on 1 protein ↔ Proteomics: higher level of information
Functions that cannot get predicted by genome sequencing:
1. Genes don’t inform about organism complexity:
Genomes: number of genes is not giving enough information about the organism complexity
o Humans: 20.000 – 40.000 genes
o Yeast: 6.000 genes
o Drosophila: 13.000 genes
o Earth worm: 18.000 genes
2. No correlation between mRNA and protein profiling:
RNA sequencing: no direct correlation between mRNA amount and protein expression
sometimes high RNA amounts give low amounts of protein / low RNA gives much proteins
3. No direct protein-gene relation:
Proteins can be post-translationally modified
Alternative splicing: 1 gene can result in different isoforms bv α -1-antitrypsin has 22 isoforms
Proteoforms: different forms of the same protein arising from 1 SNP / differential splicing / post-
translational modification
Can be linked to diseases
4. Protein interaction networks:
Protein complexes: most cellular processes regulated by protein complexes
Functional proteomics: define protein as an element in an interaction network (don’t ascribe it 1 function)
5. Cellular localisation:
Cell localisation: proteins can be localised in nucleus, cytosol, plasma membrane, mitochondria, ER
1 protein can have different functions depending on their cellular localisation
,Introduction to proteomics protocol:
1. Protein extraction from sample
2. Protein separation
3. Protein digestion with protease
4. Peptide separation by liquid chromatography
5. Electrospray ionization brings peptides in
gas phase + gives positive charge
6. Ion mobility in mass spectrometry
7. Data analysis
Bottom-up proteomics: identify proteins based on the peptide mixture
(proteome -> proteins -> enzymatic digestion -> peptide mixture)
Top-down proteomics: immediately introduce purified proteins in mass spectrometer
(proteome -> MS analysis)
Sample preparation:
1. Break up tissues/cells and extract the protein fraction
2. Modify proteins based on further analysis steps (denaturation, reduction,…)
Variables for separation success:
Method of cell lysis, type of detergent
pH
Temperature
Proteolytic degradation
Protein digestion:
LC-MS done on peptides
Trypsin: hydrolyses the C-terminal of lysine and of arginine
Chymotrypsin: hydrolyses the C-terminal of large hydrophobic residues tyrosine, phenylalanine, tryptophane
Peptide separation:
Sample enrichment: some proteins reside in the cell in lower abundancy
11 orders of magnitude between the highest and lowest abundant protein
Lower abundance proteins cannot be seen in chromassy blue staining gel
Increase peak capacity:
Sequential separation techniques: each dimension made by a technique based on different
physicochemical properties of the proteins
Separation capacity: amount of peaks fitting into chromatogram without overlapping peaks
= maximum amount of components that can be separated by the chromatographer
Peak capacity: multiplication of separation capacities
Peak capacity=CS1∗CS 2∗CSn−1∗CS n
,Chromatography:
Chromatography: separates biomolecules on the basis of their distribution over the stationary and
mobile phase
Molecules with higher affinity for the stationary phase get slowed down
The longer chromatography runs the better the separation
Ion-exchange chromatography: stationary phase in neg/pos charged
Same charge molecules go through faster
Gel permeation / filtration chromatography: small molecules get stuck in the pores
Big molecules elute faster
Reverse phase chromatography: hydrophobic parts of the protein will bind to the hydrophobic stationary phase
Polar molecules elute faster
Ion pairing reagent: has hydrophobic tail and charged head to help more polar molecules
bind the stationary phase
Reverse phase used for proteomics
SDS PAGE
Mass spectrometry:
1. Ion formation: by ionization source that converts peptides in gaseous ions
2. Ion separation: separation based on the mass/charge ratio (m/z) by electric and magnetic
fields in the mass analyzer
3. Ion detection: specific current strength produced based on m/z ratio and abundancy of the ion
Data analysis:
De novo-sequencing: determining peptide AA sequence from the spectrum
In silico spectra: experimental MS spectra compared to in silico spectra generated from a protein database
Sample preparation:
, Intact proteins:
Gel based separation
Affinity purification by antibodies
Protein chips and arrays
Antibody based identification / LC-MS/MS
Purified intact proteins analysed by LC-MS after digestion gives most detailed info
Digested proteins:
Liquid chromatography
Mass spectrometry
Proteoform info can be lost after digestion
Protein interference problem: difficult to know which proteins/ proteoforms are in a complex once
it’s digested
→ Mass spectrometry: identifies protein groups
When identifying peptide 1 and 2 : the protein in the sample can be
A/B/C/D/E
Identify A if you know peptide 0 is in it
1. Define research question:
o Investigate protein, proteoform, protein class, proteome?
o Which techniques available?
o Tertiary structures / protein complexes intact?
2. Find best sample preparation protocol:
Protocol: tailored to extract a single protein , more specific research question results in more
optimal preparation protocol
Protein diversity: bv cytosolic proteins , membrane associated proteins , protein complexes , short
open reading frames , …
General steps:
1. Experimental design
2. Literature study
3. Sample collection
4. Protein extraction , solubilization
5. Protein separation or purification
6. Reduction and alkylation
7. Digestion
8. Peptide purification or selection
9. Prefractionation
Optimal sample type:
Lab grown cells not representative for real tissue
Patient samples more variable
Serum and plasma highly variable due to protein concentration differences
