Concepts of protein technology and applications
VERGEET ARTIKEL NIET!
Chapter 1: introduction (xaveer van ostade)
1 Definition proteomics
= determination of the complete set of proteins that is present in a system under specific circumstances:
• System
o Protein complex
o Subcellular compartment
o Cell
o Tissue
o Organism
• Circumstances
o Treatment
o Time after treatment
o Condition of the cell (health, age, normal, infected, tumor…)
Proteomics: you are analyzing at that moment à changes all the time à complex. Different with genomics because
DNA does not change à easier.
2 Why proteomics?
Because many protein events cannot be predicted by genomics!
2.1 Genome sequencing and prediction of genes
Genes: contain the info, but real work happens because of proteins.
Proteins:
• Can interact
• Can be modified
• Are diverse in function à gives info on state of the cell.
Ex motorcycle: genomics = all the parts, proteomics: the parts interact with each other.
Genome sequencing: still difficult to predict genes à verification of gene product by proteomic analysis = necessary.
You can make prediction on basis of algorithms, but you need to find it expressed as a protein in a cell to say that it is
indeed a coding sequence à call it proteogenomics.
2.2 mRNA vs protein profiling
There is not always a direct correlation between mRNA expression and protein expression levels. Sometimes a little
bit of mRNA produces a lot of proteins or a lot of mRNA produces a few proteins.
2.3 More (6-8) proteins/gene
2.3.1 Posttranslational modifications (PTMs)
Proteins have posttranslational modifications and this is not given in the genome/mRNA à they appear at certain
moments in the cell à some get removed again (ex. phosphorylation). It’s a way of finetuning the activity of proteins
in the cell so it can be added or removed.
1
,2.3.2 Alternative splicing
A produced mRNA can be spliced or alternative spliced à results in other proteins from the same gene = isoforms.
2.4 Protein interaction networks
Higher order of complexity without drastic increase of number of components.
A protein almost never works on its own à they work in a complex or go to a partner protein and work together. To
know the function of a protein, you need to know with which protein they associate.
The protein you are studying is always preforming within a certain context à contextual function.
2.5 Cellular localization
Depending on the biological state of the cell, a protein can be localized in one or different cellular locations (nucleus,
cytosol, plasma membrane mitochondria, ER…).
Different binding partners in different locations à one protein can have several functions, depending on the
localization in the cell.
There are proteins who travels between cells.
à these features cannot be predicted by genome/transcript sequencing, so proteomics is necessary.
3 Proteomics as part of systems biology
To understand the dynamic complexity of an organism, an integrated image of all aspects of proteins needs to be
developed:
• mRNA and protein profiles & how these change over time (ex. during development or changing conditions
(pathological)
• Knowledge of the state and properties of all proteins:
o Posttranslational modifications
o Cellular localization
o Binding of metabolomic ligands: haem ring, metal ions, glucose, ATP, ADP, GTP, GDP…
o Alternative splicing
o Proteolytic degradation à synthesis, localization and activity status are regulating factors
o Oligomeric state & contribution in complexes
o Structure, conformation & allosteric mechanisms
• All protein-protein interactions in space and time in one cell.
Together with genomic & metabolomic data (in space and time) à systems biology
4 The different phases of proteomics
1. Proteomics sensu strictu
Large scale identification and characterization of proteins, inclusive their PTMs (‘shotgun’ proteomics).
2. Differential Proteomics
Large scale comparison of protein expression levels.
3. Cell-mapping proteomics
Protein-protein interaction studies.
2
,5 Identification of proteins: principles
5.1 Sample preparation
You can have a good mass spectrometer but if your sample is not prepared carefully, your results won’t be good!
Important to make a good design of what you’re going to do.
1. Break up tissues or cells, extract protein fraction.
2. Modification of proteins for further analysis (denaturation, reduction etc) à dependent on the forthcoming
methods for separation/purification and identification.
3. Important variables that determine the success of separation/purification and identification:
a. Method of cell lysis, type of detergent
b. pH
c. Temperature
d. Proteolytic degradation (addition of protease inhibitors)
4. In many cases: trial and error, so sometimes…
Prepare proteins for MS analysis: protease treatment (can be done before or after protein separation)
Proteases hydrolyze (specific) peptide bonds:
• Trypsin: C-terminal of Lys & Arg residues
• Chymotrypsin: C-terminal of large hydrophobic residues (Tyr, Phe, Trp)
Examenvraag van vorig jaar: ‘wat gebeurt er wanneer je chemotrypsine ipv trypsine gebruikt?’.
5.2 Protein/peptide separation
If you want to separate proteins à need to detect them à can be very difficult since the amount of cellular material
that gives you a certain amount of protein is very small (100 million cells à get 10 mg of material).
• Detect a protein with 10 protein copies/cell: you
can’t see it by gel electrophorese, Coomassie or
Silver staining.
• 1000 protein copies/cell: only see it by silver
staining.
• 100.000 protein copies: also see it by Coomassie.
à very difficult to detect low abundant proteins.
Proteomics goes into the fM and sometimes aM range.
Plasma: protein concentrations differ in concentration by 11 orders of magnitude!
There are a lot of proteins in blood à all have different abundancies. Interleukin 6 = important for
inflammation, low concentration in blood. Problem of working with blood: working with a huge over
abundancies of hemoglobin and albumin à remove before looking at low abundance proteins.
Working with complex samples: need to separate them before analyse with MS.
3
, 5.2.1 Increasing separation capacity (peak capacity)
Use sequential separation techniques (‘dimensions’) whereby each dimension is a technique based on a different
physicochemical characteristic of the proteins or peptides (orthogonal separation). Multiply separation capacities of
each dimension.
The total separation capacity can be calculated as follows:
Peak capacity = CS1 * CS2 * CSn-1 * CSn
With CSn = peak capacity for a chromatographic system n.
2D-electrophoresis (2D-page)
= combination of isoelectric focusing (1st dimension) and PAA gel electrophoresis (2nd dimension).
• Result: series of spots (with x & y coordinates), spread over the gel.
• Each spot = one or a few proteins (dependent of sample complexity).
• Separation of hundreds to thousands of proteins with determination of the pI, MW, PTM (ex. glycosylation) &
relative abundancy.
Advantages of 2D-PAGE:
• Visual
• Separation of proteins based on PTM
• Identification not always requires protein AA sequence (see below: PMF)
• Comparison of expression levels possible (most used: DIGE)
Disadvantages:
• Some proteins are difficult to separate:
o Strong basis
o Low MW
o Aggregation of some proteins at pI
• Automation is difficult
• Large sample quantities required (20-1000μg), dependent of protein abundancy and sample complexity
High pressure liquid chromatography (HPLC)
= separation of biomolecules based on their distribution over a mobile and a stationary phase.
• Mobile: proteins in solvent
• Stationary: sticked beads
à Each protein partitions itself between stationary and mobile phase
Types of chromatography:
• Ion exchange
• Gel permeation
• Reverse phase à usually used for proteomics
• Affinity
4
VERGEET ARTIKEL NIET!
Chapter 1: introduction (xaveer van ostade)
1 Definition proteomics
= determination of the complete set of proteins that is present in a system under specific circumstances:
• System
o Protein complex
o Subcellular compartment
o Cell
o Tissue
o Organism
• Circumstances
o Treatment
o Time after treatment
o Condition of the cell (health, age, normal, infected, tumor…)
Proteomics: you are analyzing at that moment à changes all the time à complex. Different with genomics because
DNA does not change à easier.
2 Why proteomics?
Because many protein events cannot be predicted by genomics!
2.1 Genome sequencing and prediction of genes
Genes: contain the info, but real work happens because of proteins.
Proteins:
• Can interact
• Can be modified
• Are diverse in function à gives info on state of the cell.
Ex motorcycle: genomics = all the parts, proteomics: the parts interact with each other.
Genome sequencing: still difficult to predict genes à verification of gene product by proteomic analysis = necessary.
You can make prediction on basis of algorithms, but you need to find it expressed as a protein in a cell to say that it is
indeed a coding sequence à call it proteogenomics.
2.2 mRNA vs protein profiling
There is not always a direct correlation between mRNA expression and protein expression levels. Sometimes a little
bit of mRNA produces a lot of proteins or a lot of mRNA produces a few proteins.
2.3 More (6-8) proteins/gene
2.3.1 Posttranslational modifications (PTMs)
Proteins have posttranslational modifications and this is not given in the genome/mRNA à they appear at certain
moments in the cell à some get removed again (ex. phosphorylation). It’s a way of finetuning the activity of proteins
in the cell so it can be added or removed.
1
,2.3.2 Alternative splicing
A produced mRNA can be spliced or alternative spliced à results in other proteins from the same gene = isoforms.
2.4 Protein interaction networks
Higher order of complexity without drastic increase of number of components.
A protein almost never works on its own à they work in a complex or go to a partner protein and work together. To
know the function of a protein, you need to know with which protein they associate.
The protein you are studying is always preforming within a certain context à contextual function.
2.5 Cellular localization
Depending on the biological state of the cell, a protein can be localized in one or different cellular locations (nucleus,
cytosol, plasma membrane mitochondria, ER…).
Different binding partners in different locations à one protein can have several functions, depending on the
localization in the cell.
There are proteins who travels between cells.
à these features cannot be predicted by genome/transcript sequencing, so proteomics is necessary.
3 Proteomics as part of systems biology
To understand the dynamic complexity of an organism, an integrated image of all aspects of proteins needs to be
developed:
• mRNA and protein profiles & how these change over time (ex. during development or changing conditions
(pathological)
• Knowledge of the state and properties of all proteins:
o Posttranslational modifications
o Cellular localization
o Binding of metabolomic ligands: haem ring, metal ions, glucose, ATP, ADP, GTP, GDP…
o Alternative splicing
o Proteolytic degradation à synthesis, localization and activity status are regulating factors
o Oligomeric state & contribution in complexes
o Structure, conformation & allosteric mechanisms
• All protein-protein interactions in space and time in one cell.
Together with genomic & metabolomic data (in space and time) à systems biology
4 The different phases of proteomics
1. Proteomics sensu strictu
Large scale identification and characterization of proteins, inclusive their PTMs (‘shotgun’ proteomics).
2. Differential Proteomics
Large scale comparison of protein expression levels.
3. Cell-mapping proteomics
Protein-protein interaction studies.
2
,5 Identification of proteins: principles
5.1 Sample preparation
You can have a good mass spectrometer but if your sample is not prepared carefully, your results won’t be good!
Important to make a good design of what you’re going to do.
1. Break up tissues or cells, extract protein fraction.
2. Modification of proteins for further analysis (denaturation, reduction etc) à dependent on the forthcoming
methods for separation/purification and identification.
3. Important variables that determine the success of separation/purification and identification:
a. Method of cell lysis, type of detergent
b. pH
c. Temperature
d. Proteolytic degradation (addition of protease inhibitors)
4. In many cases: trial and error, so sometimes…
Prepare proteins for MS analysis: protease treatment (can be done before or after protein separation)
Proteases hydrolyze (specific) peptide bonds:
• Trypsin: C-terminal of Lys & Arg residues
• Chymotrypsin: C-terminal of large hydrophobic residues (Tyr, Phe, Trp)
Examenvraag van vorig jaar: ‘wat gebeurt er wanneer je chemotrypsine ipv trypsine gebruikt?’.
5.2 Protein/peptide separation
If you want to separate proteins à need to detect them à can be very difficult since the amount of cellular material
that gives you a certain amount of protein is very small (100 million cells à get 10 mg of material).
• Detect a protein with 10 protein copies/cell: you
can’t see it by gel electrophorese, Coomassie or
Silver staining.
• 1000 protein copies/cell: only see it by silver
staining.
• 100.000 protein copies: also see it by Coomassie.
à very difficult to detect low abundant proteins.
Proteomics goes into the fM and sometimes aM range.
Plasma: protein concentrations differ in concentration by 11 orders of magnitude!
There are a lot of proteins in blood à all have different abundancies. Interleukin 6 = important for
inflammation, low concentration in blood. Problem of working with blood: working with a huge over
abundancies of hemoglobin and albumin à remove before looking at low abundance proteins.
Working with complex samples: need to separate them before analyse with MS.
3
, 5.2.1 Increasing separation capacity (peak capacity)
Use sequential separation techniques (‘dimensions’) whereby each dimension is a technique based on a different
physicochemical characteristic of the proteins or peptides (orthogonal separation). Multiply separation capacities of
each dimension.
The total separation capacity can be calculated as follows:
Peak capacity = CS1 * CS2 * CSn-1 * CSn
With CSn = peak capacity for a chromatographic system n.
2D-electrophoresis (2D-page)
= combination of isoelectric focusing (1st dimension) and PAA gel electrophoresis (2nd dimension).
• Result: series of spots (with x & y coordinates), spread over the gel.
• Each spot = one or a few proteins (dependent of sample complexity).
• Separation of hundreds to thousands of proteins with determination of the pI, MW, PTM (ex. glycosylation) &
relative abundancy.
Advantages of 2D-PAGE:
• Visual
• Separation of proteins based on PTM
• Identification not always requires protein AA sequence (see below: PMF)
• Comparison of expression levels possible (most used: DIGE)
Disadvantages:
• Some proteins are difficult to separate:
o Strong basis
o Low MW
o Aggregation of some proteins at pI
• Automation is difficult
• Large sample quantities required (20-1000μg), dependent of protein abundancy and sample complexity
High pressure liquid chromatography (HPLC)
= separation of biomolecules based on their distribution over a mobile and a stationary phase.
• Mobile: proteins in solvent
• Stationary: sticked beads
à Each protein partitions itself between stationary and mobile phase
Types of chromatography:
• Ion exchange
• Gel permeation
• Reverse phase à usually used for proteomics
• Affinity
4
