Resume
Samenvatting - Protein technology and proteome analysis (16/20 gehaald)
- Établissement
- Universiteit Antwerpen (UA)
Complete summary of this course.
[Montrer plus]
Aperçu du contenu
IntroductionProteomics = Determination of the complete set of proteins that is present in a system under specific circumstances
Different kind of What does proteomics do:
proteomics - Study the presence of proteins / amount of proteins present
- Study the posttranslational modifications
- Protein interaction networks
- Localization of the proteins
Sample prep Break tissue or cells ⇒ Extract protein fraction ⇒ Further modification of proteins like denaturation, reduction etc.
Prep for MS Protease = hydrolysis specific peptide bonds
analysis - Trypsin = Hydrolyses the C-terminal of Lys and Arg residues
- Chymotrypsin = Hydrolyses the C-terminal of large hydrophobic residues (Tyr, Phe, Trp)
Separation of Protein enrichment is needed because of the big amount of proteins present in a cell, low abundant proteins are
proteins extremely difficult to detect ↳
complex sample
➢ 1st dimension separation ⇒ 2D gel-electrophoresis, based on
○ IsoElectric Focusing (IEF)
○ Molecular weight
➢ 2nd dimension separation ⇒ HPLC, based on their affinity for a polar stationary surface such as silica,
hence it is based on analyze ability to engage in polar interaction ↳ hydrophobic
Separation: High Proteins in solvent (mobile phase) migrate through a column packed with beads (stationary phase). Each
pressure liquid protein partitions itself between stationary and mobile phase
chromatography
(HPLC) Ion exchange Is a class of liquid chromatography (LC). It separates charged biomolecules.
chromatography
bu SCX What: Proteins have a unique 3D structure and hence specific ionizable groups on their
surfaces, creating a unique surface charge at a given pH.
.
How: This feature is used to separate molecules by their interaction with oppositely charged
particles of the stationary phase. Adjusting the pH or ionic strength of the mobile phase allows
the molecules to be released from the column.
Steps:
1. Column washing ⇒ To prepare the column
2. Loading the sample ⇒ Will bind to the stationary phase with opposite charge
3. Washing ⇒ Wash with start buffer to make sure that everything that is not bound is
eluted from the column
4. Elution ⇒ Can be done in 2 ways
a. Adding a salt gradient ⇒ Salt will go in competition, the higher the charge o
the proteins the longer they stay on the column (and thus, the lower the charg
of the protein, the faster they will elute)
b. Adding a pH gradient ⇒ Proteins will come off the stationary phase when th
pH is higher/lower than the iso-electric point because then the protein will get
the opposite charge and comes off the column
poswilaugh
Gel filtration What: The column contains beads which are porous.
chromatography
How: The Pores in the beads differ in sizes, smaller proteins can enter the pore and bigger
proteins can't ⇒ Larger proteins will come out of the column first because they can't go into th
, Reverse phase Phases:
chromatography - Mobile phase = polar
- Stationary phase = non-polar = hydrophobic
·
TFA will dissolve in water into H+ and TFA-
TFA-ion:
The negative ion will bind to the positive part of amino acids ⇒ the amino acid site will now be
more hydrophobic because of the TFA-ion ⇒ the whole protein will becomes more hydrophob
H+-ion
the H+ will bind to the negative site of the protein ⇒ the whole protein will become more or les
neutral but very hydrophobic
Affinity substrate of enzyme attaches to the beads ⇒ 1 protein is then targeted by this complex
chromatography - comes handy if you want to catch 1 kind of protein or a specific family of proteins
Analysis: Separation: based on the M/Z
mass
spectrometry Mass Spec consists of 3 parts
1. Ionization source
a. ESI
b. MALDI
2. Mass analyzer
a. Magnetic sector analyzr
b. Quadrupole (usually with ESI)
c. 3D-ion trap
d. 2D-ion trap
e. Fourier-Transform ion cyclotron resonance (FT-ICR)
f. Orbitrap
g. TOF
3. Detector
a. Faraday cup
b. Electron multipliers
How it works: First the sample is gonna be ionized, the peptides are converted into ions and they are brought int
gas phase, they will then fly through the vacuum tube
Peaks: The higher the peaks, the more abundant the protein in the sample is
Identification and Principles of protein identification data analysis
analysis 1. De novo-sequencing = direct determination of the peptide AA sequence from the spectra
2. Comparing Experimental MS spectra with in silico spectra generated from protein database
- The peptide must be present in the database
Shotgun What: method used to identify and characterize proteins within a complex mixture.
proteomics
How: Breaking down the entire proteome into smaller peptide fragments through enzymatic digestion. These
fragments are then separated, typically using liquid chromatography, and analyzed by mass spectrometry to
determine their mass-to-charge ratios. By matching these spectra to protein sequence databases, shotgun
proteomics enables the identification of proteins present in the original sample.
Bottom up vs. Bottom up = When the proteins are digested into smaller fragments/peptides
, Sample preparation prior to LC-MS
Basic Proteins = molecules consisting of amino acids
knowledge Proteome = set of all proteins in a cell ⇒ but also in a organelle, cell tissue, organ etc
Proteomics = study of the proteome
Proteoforms = detection of the various forms of proteins present in a cell. Knowing the exact proteoform can be
necessary to understand diseases
ELISA and Antibody based technologies are very sensitive and recognize small epitopes. But these platforms do not acquire
LC-MS sequencing/structuur/PTM info that is connected to proteoforms ⇒ LC-MS can help to get more detailed
information
Complex Complex sample with intact proteins:
samples 1. Gel based separation like 2D-gelelectrophoresis
a. Proteoforms with different phosphorylation status can also be separated and form a
typical pattern (see figure)
2. Affinity purification with antibodies Gel based -2D-GE
3. Protein chips and arrays
4. Antibody based based identification or MS/MS after separation
Complex sample with digested proteins
1. Liquid chromatography
2. MS or MS/MS
Protein inference problem = it is VERY difficult to know which proteins or proteoforms were in a complex sample
once digested. There is information loss when you digest proteins
Mass spectrometry only identifies protein groups
Can you do MS Yes, it is possible to perform mass spectrometry (MS) and tandem mass
spectrometry (MS/MS) on intact proteins ⇒ top-down proteomics. In this
and MS/MS on
method, intact proteins are ionized and then analyzed directly in the mass
intact proteins spectrometer without prior digestion into peptides.
Top-down proteomics allows the analysis of intact proteins, providing valuable
information about post-translational modifications, sequence variants, and
structural characterization of proteins. This approach is particularly useful for
studying smaller to medium-sized proteins but may face challenges with larger
and more complex proteins due to difficulties in achieving effective fragmentation
and analyzing the resulting spectra.
Sample prep There are different steps in sample prep, see next columns
steps:
1. Define your Do I want to investigate a protein, proteoform, a protein class, RNA-binding proteins, the whole proteome? Do I
research want to keep the tertiary structures? ⇒ Based on this you’re gonna choose a research method
question
2. Prepare / find 1. Make up the scientific question
the correct 2. Literature study
protocol for 3. Sample collection ⇒ stabilize the sample ASAP otherwise degradation will happen
sample prep 4. Protein extraction and solubilization
5. Optional: Protein separation or purification => often for complex samples
6. Reduction and alkylation
7. Digestion
8. Peptides purification or selection
9. Optional: Pre-fractionation
Sample types
- Fresh is best ⇒ Proteins degrade quickly!
- Cell lines can be grown under controlled settings, but not always representative for real tissue/ cells
- Patient samples, serum and plasma can be highly variable
3. Sample What causes protein degradation:
degradation has ➢ Stress
to be minimized ○ can reduce proteolytic activity in the cell
○ can induce phosphatase activity ⇒ phosphorylated proteins can disappear within minutes (so is a
problem!)
, 4. Protein 1. Disrupt the cell membrane to bring proteins in solution, how?
extraction Mechanical vs non-mechanical
a. Mechanical ⇒ b. Non-mechanical ⇒
i. sonification i. detergents
ii. mixing ii. chaotropic agents
iii. freeze thawing
iv. bead beater
Soft vs Harsh ⇒ Can induce protein denaturation
a. Soft ⇒ b. Harsh ⇒
i. Osmotic shock i. Blender
ii. Detergents ii. Tissue chopper
iii. Enzymatic digestion iii. Cyro-grinding = Grinding with mortar under liquid N2
iv. Dounce homogenizer iv. Bead beater
v. Sonication
!! prevent protein degradation by adding inhibitors for example
After disruption of the cell membrane, you will end up with a sample with proteins in a solution and remnants of
cells
2. Subcellular fractionation with centrifugation
a. Differential centrifugation
i. 5000g for10 min ⇒ really slow, pellet with nuclear fraction
ii. Then you do 10.000g for 20 min ⇒ Pellet of mitochondria
iii. Then you do 100.000g for 1h ⇒ pellet with the rest of the membrane
b. Sucrose gradient
3. Protein extraction and solubilization
- Can be done with a lysis buffer
- you need to think about whether the proteins need to denature or not
Chaotropic agents Urea (7-8M)
⇒ Denature proteins by disrupting non-covalent - Good for 2D-PAGE
bonds - Efficient with H-bond disruption
⇒ Change hydrogen bonding in the solvent - Poor for hydrophobic disruption
⇒ Disrupt the hydrogen bonds and hydrophobic
interactions between and within the proteins leading to Thiourea (2M)
denaturation - Good for hydrophobic disruption
Guanidinium chloride (6M)
- Good for H-bond disruption
- Good for hydrophobic region disruption
Detergents SDS (anionic)
⇒ has hydrophobic and hydrophilic region - Denaturing
⇒ the hydrophobic core associates with hydrophobic - Break protein-protein interaction
regions of of proteins
⇒ Used to break hydrophobic interactions and Triton* X100 & Tween (non-ionic)
increase solubility of proteins - Used when critical to maintain protein function
- Break lipid-lipid and lipid-protein interactions but NOT
protein-protein ⇒ non-denaturing
CHAPS (Zwitterionic detergents)
- disrupt protein-protein interactions
- Used for IEF and ion-exchange chromatography
4. Detergent etc. removal
Detergents, salts, protease inhibitors etc. need to be removed prior to LC-MS or other steps ⇒ they interfere with
analysis,
On protein level this is done by:
- Dialysis
- Gel filtration chromatography
- Cutoff filters
- SDS-PAGE
- Protein precipitation = acetone precipitation, most widely used way
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 yarameijs2001. Stuvia facilite les paiements au vendeur.
Est-ce que j'aurai un abonnement?
Non, vous n'achetez ce résumé que pour €15,86. Vous n'êtes lié à rien après votre achat.
Peut-on faire confiance à Stuvia ?
4.6 étoiles sur Google & Trustpilot (+1000 avis)
56326 résumés ont été vendus ces 30 derniers jours
Fondée en 2010, la référence pour acheter des résumés depuis déjà 14 ans