Concepts of Protein technology and applications (2050FBDBMW)
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Summary Concepts of Protein technology and applications (18/20)
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Concepts of Protein technology and applications (2050FBDBMW)
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Universiteit Antwerpen (UA)
This large summary covers all chapters of the course Concepts of Protein Technology and Applications, taught by Professors Van Ostade and Boonen. It is based on PowerPoint slides and lecture notes, providing a comprehensive overview of the subject matter.
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Concepts of Protein technology and applications (2050FBDBMW)
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HOOFDSTUK 1 : INTRODUCTION
1.1 DEFINITION
1.1.1 PROTEOMICS
Proteomics = determination of the complete set of proteins that is present in a system under specific
circumstances
System Circumstances
● Protein complex ● Treatment
● Subcellular compartment ● Time after treatment
● Cell ● Condition of the cell (age, normal, infected, tumor,..)
● Tissue
● Organisms (yeast, drosophila,..)
1.2 WHY PROTEOMICS
OVERVIEW
Why proteomics
1) Workhorses of the cell
2) Genome sequencing
3) mRNA vs protein profiling
4) 1 gene encodes for multiple (6-8) proteins
5) Protein interaction networks
6) Cellular localization
All these features cannot be predicted by genome sequencing → need proteomics!
,1.2.1 WORKHORSES OF THE CELL
Proteins = are the workhorses of the cell : the real thing
Example : motorcycle
● Genomics = the genes are a list of all the parts that you need for a motorcycle
● Proteomics = the proteins are the actual parts of the motorcycle that work together
1.2.2 GENOME SEQUENCING
Genome sequencing = the determination of the genes
● Sometimes it is still difficult to predict genes (where is the start and stop codon,...)
● Verification of the gene product by proteomic analysis is still necessary
○ Based on the amino acid sequence of the protein we can look at the gene sequence =
proteogenomics
1.2.3 MRNA VS PROTEIN PROFILING
No direct correlation = between mRNA expression level and protein expression level
● Some cells have high levels of mRNA but low levels of protein expression
● Some cells have low levels of mRNA but high levels of protein expression
1.2.4 ONE GENE ENCODES FOR MORE PROTEINS (6-8)
Concepts = 1 gene can produce multiple proteins → on average 6-8
Due to :
1) Posttranslational modifications (PTM’s)
2) Alternative splicing (isoforms)
➔ These features cannot be predicted by genome sequencing
➔ Therefor we need proteomics
1.2.5 PROTEIN INTERACTION NETWORKS
Protein complexes = most cellular processes are regulated by protein complexes instead of individual proteins
● Genes do not interact with each other → proteins have too : to lead to a function
● These interactions can only be studies by proteomics
1.2.6 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, ER,..). Different locations mean different binding partners → one protein
can have several functions, depending on the localisation in the cell.
,1.3 PROTEOMICS AS A PART OF SYSTEM BIOLOGY
1.3.1 WHAT IS SYSTEM BIOLOGY
System biology = bringing all the info of different levels together (metabolomics, genomics, proteomics) to
have a global view on the system
Goal = when a cell is under certain circumstances, we want to know what events are happening in the cell
1.3.2 WHAT DO WE NEED TO KNOW FOR SYSTEM BIOLOGY
In order to understand the dynamic complexity of an organism, an integrated image of all aspects of proteins
needs to be developed (so far only the average of all possible states is measured):
1) mRNA and protein profiles → how they change over time = during development /changing conditions
2) Protein-protein interactions in space and time
3) Knowledge of the state and properties of all proteins :
● Posttranslational modifications
● Cellular localization
● Binding of ‘metabolomic’ ligands
● Alternative splicing
● Proteolytic degradation
● Oligomeric state and contribution in complexes
● Structure, conformation and allosteric mechanisms
Together with genomic and metabolomic data (in space and time) → systems biology
1.4 THE DIFFERENT SORTS OF PROTEOMICS
Proteomics sensu strictu Large scale identification and characterization of proteins, inclusive their
posttranslational modifications
Differential proteomics Large scale comparison of protein expression levels
Example
● Expression of proteins in healthy and cancerous tissue
● Controlling a higher or lower expression of proteins
Sample preparation
1) Break up tissues or cells
2) Extract protein fraction
3) Modification of proteins for further analysis (denaturation, reduction,..)
Variables = that determine the success of separation / purification and identification
● Method of cell lysis, type of detergent
● pH
● Temperature
● Proteolytic degradation (addition of protease inhibitors)
PREPARE PROTEINS FOR MASS SPECTROMETRY ANALYSIS
Protease treatment = to prepare proteins for MS analysis
● Can be done before or after protein separation
● Proteases hydrolyse peptide bonds → proteins break into small peptides
● Advantage : mass spectrometer works better with peptides
Examples
1) Trypsine : cuts at C-terminal of Lys and Arg residues
2) Chymotrypsin : cuts at C-terminal of large hydrophobic residues (Tyr, Phe, Trp)
→ cutting on specific places!
, 1.5.2 SEPARATION OF PROTEINS / PEPTIDES
If you want to separate proteins you need to detect them, which
can be very difficult since the amount of cellular material that
gives you a certain amount of protein is very small.
Example
If you use 100 million cells (1.10^8) you get 10 mg of material.
If you want to detect a protein with 10 protein copies/cell you
can’t see it by gel electrophoresis, Coomassie or Silver staining.
Example
With 1000 protein copies/cell you will only see it by silver staining, if there are 100.000 protein copies you can
see it also by Coomassie. It is thus very difficult to detect low abundant proteins.
➔ Proteomics goes into the fM and sometimes aM range.
PROTEIN CONCENTRATIONS DIFFER IN 1 SAMPLE
Example : plasma sample
● Different proteins present in different amounts
In plasma the protein concentrations vary by 11 orders of magnitude.
You have very abundant proteins like hemoglobin and albumin, but you
have proteins that are very low abundant, like interleukins.
INCREASING SEPARATION CAPACITY (PEAK CAPACITY)
Sequential separation techniques = the use of sequential separation techniques (‘dimensions’) whereby each
dimension is a technique based on a different physicochemical characteristic of the proteins or peptides
(orthogonal separation)
To separate proteins, you can use several separation methods. Every separation method has a peak capacity:
the maximum amount of peaks you can have in a certain separation.
By using two methods that make use of different physical or chemical characteristics you use orthogonal
methods. This enables you to multiple the peak capacity of both methods.
Figure: 1st dimension is separation based on charge.
2nd dimension is based on physicochemical
characteristics.
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