This summary contains notes taken during the courses and all information needed to pass the exam (of both professors Van Ostade and Boonen) and information from the slides. Summary and elaboration of all exam questions of the course Concepts of Protein technology and applications (17/20): This is a...
Partim Boonen: Concepts of Protein
technology and applications
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Concepts of Protein Technology &
Applications: partim Boonen
Sample preparation for proteome analysis
Proteins = molecules consisting of AA that perform much of life’s function
Proteome = set of all proteins in a cell
Proteomics = study of the structure and function of proteins
Proteoform = any of several different forms of the same protein, arising from either SNPs, differential
splicing of mRNA, or post-translational modifications (phosphorylation, glycosylation..).
Detection of the various forms of proteins present in a cell or causing diseases requires analytical
techniques with high precision and sensitivity. Knowing the exact proteoforms can be necessary to
understand diseases. This is often done by mass spectrometry.
Issues when providing protein samples
1. Complex samples
When you have a complex sample, there are different pathways you can follow.
1. You can leave the proteins intact.
Gel-based separation
Affinity purification by antibodies
Protein chips and arrays for multiplexing
AB based identification or LCMS/MS
after separation
Edman degradation after purification
2. You can work with digested proteins
Liquid chromatography
Mass spectrometry based identification
Usually there is done a separation step before
the MS step BUT it is very difficult to know which proteins and/or proteoforms were in a complex sample once
digested. This is called the protein inference problem. First of all, you don’t detect all peptides from the original
proteoforms. If we have a lot of peptides, it’s possible proteins have isoforms or related sequences, or it’s
possible to have mutations.. It’s very difficult to know from which protein the peptides are. It’s possible you lose
information crucial for your research questions.
LC-MS/MS identifies protein families (see further) but interpretation can be difficult.
2. Diversity of proteins
Protocols for DNA and RNA are often not as diverse as sample preparations for proteins. DNA and RNA have a
polar backbone and are soluble in water but proteins can have very different chemical and physical properties,
dependent on which AA are present. This depends on charge, hydrophobicity, … Also the length of the protein is
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a big factor and if you want short reading frames or long reading frames. Proteins can also form complexes. A
more specific research question will result in a more optimal preparation protocol.
Sample
Optional preparation: general
Not optional steps
1. Scientific question – experimental design
2. Literature study
3. Sample collection 7. Digestion
4. Protein extraction – solubilization 8. Peptides purification or selection
5. Protein separation or purification 9. Prefractionation: separate complex sample into smaller
6. Reduction and alkylation samples.
Cell lines can be grown under controlled laboratory settings but are not always representative of real
tissues/cells. Patient samples are more variable due to differences in handling and patient differences. Large
sample numbers are needed for statistical good results. Serum & plasma can be variable and hard to analyse due
to large differences in protein conc. Bio-banked samples can be very variable, especially Formalin-Fixed Paraffin
Embedded tissues.
It’s important to stabilize your sample as quick as possible by putting in on ice and adding protease inhibitors.
Proteases are enzymes present in mammalian cells that digest the sample. You have to get rid of the inhibitors
again before putting them in the MS. This also counts for phosphatases: phosphatase inhibitors also often
added.
Sample degradation has to be to be minimized, especially in cells, tissues and organs of higher organisms.
- Stress can induce proteolytic activity in the cell
- Stress induces phosphatase activity: phosphorylated peptides can disappear within minutes.
- Proteins are not as stable as DNA. The stability of proteins varies greatly.
Protein extraction
= Disrupt cell membrane (and cell wall if present) to bring as many proteins in solution as possible (completeness
can’t be achieved). There are different methods for different samples. This extraction can be done mechanically:
sonification, bead beating, mixing,… Or non-mechanical. Some soft techniques keep protein structure, so you
can extract protein complexes. You have to prevent protein degradation by adding a complete protease inhibitor
mix.
Soft methods:
If your cells don’t have a rigid cell walls you can collapse the cell walls by freezing the cells and thus get a lot of
proteins out. This is a soft method called osmotic shock.
With detergents, it’s possible to make holes in the cell membrane so proteins can get out. Depending on the
detergent, it can be soft or harsh. Some pull the protein completely inside out but others leave them intact.
If there is one, you can try to digest the cell wall by lysozyme to break down peptidoglycan of gram-pos bacteria.
A dounce homogenizer is a glass tube with a small clearance between the head and tube. The cells are in a liquid
and are forced in between the tube and the homogenizer which causes friction to break the cells (minimal
heating) You want to keep certain organelles, like the nucleus intact so the exact clearance is important. This is a
soft way.
Harsh methods:
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