Resume
Summary of the course: concepts of protein technology
- Établissement
- Universiteit Antwerpen (UA)
This summary contains notes taken during the courses (of both professors Van Ostade and Boonen) and information from the slides.
[Montrer plus]
Vendeur
S'abonnerAperçu du contenu
SAMENVATTING CONCEPTS OFPROTEIN TECHNOLOGY AND
APPLICATIONS-X.OSTAEDE
Universiteit Antwerpen
Joke Stynen
Course info
• 25 hrs theory
• Read an article:
o You need to understand the workflow that the authors used to
perform their research
o Recent article
o Question about this on the exam
• Examination: Written
o – ± 5 questions (can have subquestions):
▪ 2 questions (Prof. Dewilde/Maudsley), may include
an exercise (15 points)
▪ 2 questions (Prof. Van Ostade), may include an
exercise (15 points)
▪ 1 question: article (10 points) !!
o Not successful in part prof. Maudsley or Van Ostade (< 8/20) -
> no credits !!
• STUDY THE SLIDES!! (from bb)
• Recordings from last year and link to recordings
, CONCEPTS OF PROTEIN TECHNOLOGY AND
APPLICATIONS: PARTIM X. OSTAEDE
H1: PROTEOMICS: INTRODUCTION
1. DEFINITION OF PROTEOMICS
• Determination of the complete set of proteins that is present in a system, under specific
circumstances:
o System:
▪ Proteïn complex (large or small)
▪ Subcellular compartment: organelles (mitochondria, nucleus…)
▪ Cell
▪ Tissue
▪ Organism (yeast cell, drosophila, but not a human being because this is to
complex)
o Circumstances:
▪ Treatment
▪ Time after treatment
▪ Condition of the cell (age, normal, infected, tumor…)
▪ …
• Proteomics: a dynamic system that changes all the time!
2. REASONS FOR PROTEOMICS
Because many protein events cannot be predicted by genomics!
2.1. GENOME SEQUENCING AND PREDICTION OF GENES
COMPARED TO GENOMICS, PROTEOMICS IS “THE REAL THING”
• Cell consist of different genes which can be sequenced
o These gene contain information
• For the actual situation of the cell → proteomics is needed
o Proteins are diverse in there function which gives information on the state of the cell
o Proteins can change, interact, be modified…
• Example: a motorcycle cycle and you know all the parts → genomics; assembly and
interaction of these parts → proteomics
GENOME SEQUENCING
• Humans: ± 20-40.000 genes, yeast 6000, Drosophila 13000, Ceanorhabditis 18.000, plant
26.000
o New genes are found all the time but the algorithms sometimes miss genes
• Still difficult to predict genes: verification of gene product by proteomic analysis is still
neccessary
“Proteogenomics”
1
,2.2. MRNA VS. PROTEIN PROFILING
• mRNA sequencing gives information on expression
levels of mRNA
• But the level of mRNA doesn’t always reflect the
level of proteins
o Not always a direct correlation between
expressed amount of mRNA and abundancy
of that protein → microarrays are
insufficient to measure protein expression
o Little mRNA can produce a lot of protein and vice versa
2.3. MORE (6-8) PROTEINS/GENE PROTEIN
POST TRANSLATIONAL MODIFICATIONS (OF PROTEINS)
• They appear at certain moments in
the cell
• This is a way of finetuning the activity
of proteins in the cell and
o Therefore it can be added
and removed
ALTERNATIVE SPLICING -> ISOFROMS
• Alternative splicing can result in different proteins
from the same gene
o Parts are similar but other parts have
different sequences
• Example: a-1-antitrypsin can be spliced into 22
different forms!
2.4. PROTEIN INTERACTION NETWORKS
• Proteins almost never work alone
o To know the function we need to know the partners with which it associates
o So contextual function of a protein
• Most cellular processes are regulated by protein complexes instead of individual proteins
o This results in a higher order of complexity without
drastic increase of number of components
o About 78% of yeast proteins is involved in complex
• Functional proteomics: definition of protein as an
element in an interaction network (‘contextual function’),
rather than ascribing to one function.
2
,2.5. CELLULAR LOCALIZATION
• Depending on the biological state of the cell, a
protein can be localised in one or different cellular
locations (nucleus, cytosol, plasma membrane
mitochondria, ER…).
• Different binding partners in different locations →
o One protein can have several functions,
depending on the localisation in the cell.
ALL THESE FEATURES CANNOT BE PREDICTED BY GENOME SEQUENCING → PROTEOMICS
3. PROTEOMICS AS PART OF SYSTEMS BIOLOGY
• Proteins can change all the time
o We must perform several studies and integrate the result to have a realistic idea of
the behaviour of the proteome in the cell
• To understand the dynamic complexity of an organism → 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 and how these change over time, e.g. during
development or changing conditions (e.g. pathological).
2. Knowledge of the state and properties of all proteins:
▪ Posttranslational modifications
▪ Cellular localization
▪ Binding of ‘metabolomic’ ligands: e.g.. haem ring, metal ions, glucose, ATP,
ADP, GTP, GDP… .
▪ Alternative splicing
▪ Proteolytic degradation . Hence, synthesis, localization and activity status of
a protease are regulating factors.
▪ Oligomeric state and contribution in complexes.
▪ Structure, conformation and allosteric mechanisms
3. All protein-protein interactions in space and time in one cell
• Together with genomic and metabolomic data (in space and time) -> systems biology
4. THE DIFFERENT FACES OF PROTEOMICS
• Proteomics sensu strictu *
o Large scale identification and characterization of proteins, inclusive their
posttranslational modifications.
• Differential Proteomics *
o Large scale comparison of protein expression levels.
• Cell-mapping proteomics
o Protein-protein interaction studies
* Are being dealt with in this course
3
,5. IDENTIFICATION OF PROTEINS: PRINCIPLES
• Bioinformatics to identify and quantify the proteins
5.1. SAMPLE PREPARATION
• Sample preparation is very important!!
o Garbage in → garbage out, no matter how good your instrument (mass
spectrometer) is
1. Break up tissues or cells, extract protein fraction.
2. Modification of proteins for further analysis (denaturation, reduction etc. )
o Dependent on the forthcoming methods for separation/purification and
identification
3. Important variables that determine the success of separation/purification and identification:
o Method of cell lysis, type of o Temperature
detergent o Proteolytic degradation (addition of
o pH protease inhibitors).
4. In many cases: trial and error
• It’s needed to prepare proteins for MS analysis
o This is done by a protease treatment (before or after protein separation) → proteins
will be cut into peptides (smaller fragments)
• Proteases cut proteins into peptides by hydrolysing (specific) peptide bonds:
o Trypsin: C-terminal of Lys and Arg residues
▪ Av. Length will be around 14 az
o Chymotrypsin: C-terminal of large hydrophobic residues (Tyr, Phe, Trp)
▪ You get larger peptides!
5.2. SEPARATION OF PROTEINS AND PEPTIDES
• For the separation of proteins, we need to be able to detect these proteins
• The dynamic range of a protein in a cell
is very wide: between 10 en 1.000.000
copies/cel
o It’s extremely difficult to detect
low abundant proteins
o Protein enrichment is therefore
necessary in cases of complex
samples!
4
, • (Blood)plasma: protein concentrations differ in by 11 orders of magnitude!
o Problem: there is a huge overabundancy of a lot of proteins →these need to be
removed before studying the lower abundance proteins
INCREASING SEPERATION CAPACITY (PEAK CAPACITY)
• Separation capacity = peak capacity = maximum amount of peaks there can be in a certain
separation
• Peak capacity can be increased by using several dimensions = orthogonal separation
o Use sequential separation techniques (“dimensions”) whereby
o Each dimension is a technique based on a different physicochemical characteristic of
the proteins or peptides
• This multiplies the separation capacities of each dimension
o Mostly 2D separation is used multiple times
• The total separation capacity can be calculated as follows:
Peak capacity = CS1 X CS2 X CSn-1 X CSn
With CSn = Peak capacity for a chromatographic system n
2D-ELECTROPHORESIS (2D-PAGE)
• Combination of isoeletric focusing (dimension 1)
and PAA-gelelectrophoresis (dimension2)
o Separation on charge and MW
• It gives a series of spots, spread over the gel
• Each spot is one or a few proteins
o Dependent of sample complexity
• Separation of hundreds to thousands of proteins
with determination of the pI, MW, PTM (e.g.
glycosylation) and relative abundancy.
CHROMATOGRAPHY
• Separation of biomolecules on the basis of their distribution over a mobile and a stationary
phase
• Principle of High Pressure Liquid Chromatography
(HPLC)
o Proteins in solvent (mobile phase) migrate
through column, packed with beads
(stationary phase)
o Each protein partitions itself between
stationary and mobile phase
5
Les avantages d'acheter des résumés chez Stuvia:
Qualité garantie par les avis des clients
Les clients de Stuvia ont évalués plus de 700 000 résumés. C'est comme ça que vous savez que vous achetez les meilleurs documents.
L’achat facile et rapide
Vous pouvez payer rapidement avec iDeal, carte de crédit ou Stuvia-crédit pour les résumés. Il n'y a pas d'adhésion nécessaire.
Focus sur l’essentiel
Vos camarades écrivent eux-mêmes les notes d’étude, c’est pourquoi les documents sont toujours fiables et à jour. Cela garantit que vous arrivez rapidement au coeur du matériel.
Foire aux questions
Qu'est-ce que j'obtiens en achetant ce document ?
Vous obtenez un PDF, disponible immédiatement après votre achat. Le document acheté est accessible à tout moment, n'importe où et indéfiniment via votre profil.
Garantie de remboursement : comment ça marche ?
Notre garantie de satisfaction garantit que vous trouverez toujours un document d'étude qui vous convient. Vous remplissez un formulaire et notre équipe du service client s'occupe du reste.
Auprès de qui est-ce que j'achète ce résumé ?
Stuvia est une place de marché. Alors, vous n'achetez donc pas ce document chez nous, mais auprès du vendeur jokestynen. Stuvia facilite les paiements au vendeur.
Est-ce que j'aurai un abonnement?
Non, vous n'achetez ce résumé que pour €5,99. Vous n'êtes lié à rien après votre achat.
Peut-on faire confiance à Stuvia ?
4.6 étoiles sur Google & Trustpilot (+1000 avis)
66579 résumés ont été vendus ces 30 derniers jours
Fondée en 2010, la référence pour acheter des résumés depuis déjà 14 ans