L5: Special Topic:
Quantification of protein
concentrations
DATE Feb 26, 2021
DONE
LECTURER Roland Brock
LEVEL
Revise 1
Revise 2
Revision dates
Revision documents
SLIDES Brock-Protein-Concentrations-2021.pdf
WEEK WEEK 5
Word count 2065
Table of contents
Table of contents
Lecture
General considerations concerning protein concentrations
Determination of protein concentrations (relative/absolute)
In cell lysates
Lecture
General considerations concerning protein
concentrations
Why are protein concentrations Michaelis Menten:
L5 Special Topic: Quantification of protein concentrations 1
, important? The rate of the enzymatic
reactions depends on the
It's chemistry
substrate concentration.
The law of mass action governs
The substrate turn-over depends
complex formation
on enzyme concentration
The dissociation constant = A x
B / AB
Substrate turn-over depends on
enzyme concentration (under
limiting concentration)
The simple concern that overexpression of a protein, can lead to artefacts in
signal transduction
A direct reflection of the laws of mass action
Determination of protein concentrations
(relative/absolute)
Determination of protein concentrations is pretty challenging
Since proteins are translated from mRNA, why not determine mRNA level by
qPCR?
qPCR gives relative information and not absolute about the number of
molecules
→ mRNA expression only poorly correlates with protein concentration.
→ the correlation coefficients are rather low for mRNA & protein
concentration.
Can we at least say that for certain complexes or pathways there are better
correlations?
For pathway-specific correlation the correlation is somewhat higher
Involved in metabolic and signalling pathways
But still mRNA cannot serve as reliable predictor for protein
concentrations.
For a proteasome, there is not a strong correlation
And other protein complexes
L5 Special Topic: Quantification of protein concentrations 2
, Another challenge for protein concentrations:
Protein concentrations cover an enormous dynamic range
Reaches over 5 order of magnitude
From 41 to 1.3 million proteins/yeast cell
This makes it harder to make quantitative conclusions
Even within a metabolic pathway: protein concentrations might vary
significantly.
Proteins of the glycolysis citric acid cycle
Along glycolysis, rather high concentrations
When you go deeper in the citric acid cycle, you notice a more broad
range
Example: differences in copy numbers
If you think about the whole entity of the genes present
Only the quantitaive proteome really presents the quantitive composition of
a cell.
The genome has information for different situations (cell differentation)
Only a part of the genome is expressed at a point in time.
There are a lot of different copy numbers of all the genes and therefore
everyone understands that only when it's considered on a quantitative level,
you get a good understanding of the full picture.
If you look further in the cell, there are different levels of complexity (in local
concentrations, e.g. plasma membrane)
Post-translational modifications
Phosphorylation
Ubiquitination
L5 Special Topic: Quantification of protein concentrations 3
, Subcellular protein concentrations
→ when a local concentration increases enough, it can be sufficient to drive
the reaction.
Why is protein quantification fundamentally different from oligonucleotide
quantification?
Oligonucelotides: information encoded in sequence of 4 identical building
blocks
Oligonucleotides are chemically nearly identical
Signals depend on length/amount of oligonucleotides
Proteins/peptides: 20 building blocks with very different chemical
characteristics
Proteins are chemically quite different
Signals are protein dependent
Quantification always needs a reference
Which amino acid are the most relevant in protein concentrations?
Aromatic residues
If you know the primary sequence you can determine the extinction
coefficient.
Quantitative aspects in protein detection (i)
Coomassie brilliant blue
Binds non-covalently to proteins (hydrophobic pockets/positively
charged residues
BGG bovine gamma globuline / BSA bovine serum albumin
Also for the same weight, staining intensity differs.
How different proteins are, and how different they can respond to
different stains.
→ this would not be the case for oligonucleotides
L5 Special Topic: Quantification of protein concentrations 4
Quantification of protein
concentrations
DATE Feb 26, 2021
DONE
LECTURER Roland Brock
LEVEL
Revise 1
Revise 2
Revision dates
Revision documents
SLIDES Brock-Protein-Concentrations-2021.pdf
WEEK WEEK 5
Word count 2065
Table of contents
Table of contents
Lecture
General considerations concerning protein concentrations
Determination of protein concentrations (relative/absolute)
In cell lysates
Lecture
General considerations concerning protein
concentrations
Why are protein concentrations Michaelis Menten:
L5 Special Topic: Quantification of protein concentrations 1
, important? The rate of the enzymatic
reactions depends on the
It's chemistry
substrate concentration.
The law of mass action governs
The substrate turn-over depends
complex formation
on enzyme concentration
The dissociation constant = A x
B / AB
Substrate turn-over depends on
enzyme concentration (under
limiting concentration)
The simple concern that overexpression of a protein, can lead to artefacts in
signal transduction
A direct reflection of the laws of mass action
Determination of protein concentrations
(relative/absolute)
Determination of protein concentrations is pretty challenging
Since proteins are translated from mRNA, why not determine mRNA level by
qPCR?
qPCR gives relative information and not absolute about the number of
molecules
→ mRNA expression only poorly correlates with protein concentration.
→ the correlation coefficients are rather low for mRNA & protein
concentration.
Can we at least say that for certain complexes or pathways there are better
correlations?
For pathway-specific correlation the correlation is somewhat higher
Involved in metabolic and signalling pathways
But still mRNA cannot serve as reliable predictor for protein
concentrations.
For a proteasome, there is not a strong correlation
And other protein complexes
L5 Special Topic: Quantification of protein concentrations 2
, Another challenge for protein concentrations:
Protein concentrations cover an enormous dynamic range
Reaches over 5 order of magnitude
From 41 to 1.3 million proteins/yeast cell
This makes it harder to make quantitative conclusions
Even within a metabolic pathway: protein concentrations might vary
significantly.
Proteins of the glycolysis citric acid cycle
Along glycolysis, rather high concentrations
When you go deeper in the citric acid cycle, you notice a more broad
range
Example: differences in copy numbers
If you think about the whole entity of the genes present
Only the quantitaive proteome really presents the quantitive composition of
a cell.
The genome has information for different situations (cell differentation)
Only a part of the genome is expressed at a point in time.
There are a lot of different copy numbers of all the genes and therefore
everyone understands that only when it's considered on a quantitative level,
you get a good understanding of the full picture.
If you look further in the cell, there are different levels of complexity (in local
concentrations, e.g. plasma membrane)
Post-translational modifications
Phosphorylation
Ubiquitination
L5 Special Topic: Quantification of protein concentrations 3
, Subcellular protein concentrations
→ when a local concentration increases enough, it can be sufficient to drive
the reaction.
Why is protein quantification fundamentally different from oligonucleotide
quantification?
Oligonucelotides: information encoded in sequence of 4 identical building
blocks
Oligonucleotides are chemically nearly identical
Signals depend on length/amount of oligonucleotides
Proteins/peptides: 20 building blocks with very different chemical
characteristics
Proteins are chemically quite different
Signals are protein dependent
Quantification always needs a reference
Which amino acid are the most relevant in protein concentrations?
Aromatic residues
If you know the primary sequence you can determine the extinction
coefficient.
Quantitative aspects in protein detection (i)
Coomassie brilliant blue
Binds non-covalently to proteins (hydrophobic pockets/positively
charged residues
BGG bovine gamma globuline / BSA bovine serum albumin
Also for the same weight, staining intensity differs.
How different proteins are, and how different they can respond to
different stains.
→ this would not be the case for oligonucleotides
L5 Special Topic: Quantification of protein concentrations 4
