This summary contains a detailed but clear overview of the course ‘Advanced Protein technology and proteome analysis’ taught by Prof Boonen and Van Ostade. The lessons/slides are often difficult to understand, making the extra information useful. This summary also features a newly added chapter...
ADVANCED PROTEIN
TECHNOLOGY AND
PROTEOME ANALYSIS
1st Master Biomedical Sciences
Moleculaire mechanismen van ziekten
Gedoceerd door: X. Van Ostade en K. Boonen
UAntwerpen
,H1: Post translational modifications (PTMs)
PTM’s are very important à proteomics can be used to detect PTM’s on proteins!
1. CYSTINE BRIDGE FORMATION (= FIRST PTM)
Cys$ne bridges (= disulfide bonds): common PTM
• Crucial role in stabiliza@on of protein structures à vital for maintaining conforma@ons and func@on
• Bonds form between the sulfur (-S) atoms of two cysteine residues within a protein
Difference between (–SH)₂ and –S–S–
• (–SH)₂: Refers to the presence of two thiol (-SH) groups, one on each cysteine residue.
• –S–S–: Represents the disulfide bond (cys@ne), where the two thiol groups from different cysteine residues
have undergone an oxida@on reac@on to form a covalent linkage.
• The mass difference between free cysteine (–SH) and cys@ne (–S–S–) is 2 Da.
o This difference between free thiol groups and disulfide bonds is small, making it challenging to detect
without high-resolu$on mass spectrometry.
Deriva$za$on of Free SulEydryl Groups:
• Reduc$on of the protein with agents like β-mercaptoethanol breaks disulfide bonds, resul@ng in free
sulPydryl groups.
• Before and aQer reduc@on, free sulPydryl groups (–SH) can be chemically modified.
• Para-hydroxy mercury benzoate (pHMB) is one such chemical reagent used for deriva@za@on
o Adding a mass shiQ of 321 Da.
• Deriva$za$on with pHMB or similar reagents before and aQer reduc@on allows for the iden@fica@on of
cysteines involved in disulfide bridge forma@on.
AQer deriva@za@on and trypsin diges@on (to break the protein into smaller pep@des), mass spectrometry sequencing
can reveal which cysteines were originally involved in forming the disulfide bonds.
• The modifica@on and resul@ng mass shiQ (e.g., 321 Da from pHMB deriva@za@on) enable the specific
detec@on of cysteine residues par@cipa@ng in disulfide bonding.
• The exact posi@ons of the cysteines can be determined by analyzing the mass shiQ paWern in the mass
spectrometry data, correla@ng it with the known pep@de sequences.
2. PHOSPHOPROTEOMICS (= SECOND PTM)
2.1. GENERAL
Phosphoproteomics
• Subfield of proteomics
• Focused on the iden@fica@on, quan@fica@on, and analysis of phosphorylated proteins and their
phosphoryla@on sites.
• Plays a vital role in understanding signaling pathways and cellular regula@on because phosphoryla@on is a
key post-transla@onal modifica@on (PTM) involved in many biological processes.
1
,Prevalence and complexity
• High occurrence: It is es@mated that > 50% of all proteins are phosphorylated at least once during their
life@me, with >100,000 phosphoryla@on sites across the proteome.
• Combinatorial complexity: Many proteins can have mul@ple phosphoryla@on sites, leading to diverse
phosphoryla@on combina@ons (e.g., singly, doubly, or mul@ply phosphorylated states).
Key challenges
• Low abundance and stoichiometry of phosphorylated proteins
o OQen only a small frac@on of the protein is phosphorylated à problem with sensi@vity (detec@on)
• Dynamic regula@on of phosphoryla@on
o Phosphoryla@on is reversible and @ghtly regulated by kinases/phosphatases
o Phosphoryla@on paWerns can change rapidly in response to s@muli
• Ioniza@on suppression
o Nega@ve charge of phosphate groups can reduce ioniza@on efficiency of MS
o This leads to low signal intensi@es
Workflow
1. Enrichment: Techniques to isolate phosphorylated pep@des.
2. Mass Spectrometry: Advanced methods (e.g., ETD, CID) iden@fy and localize phosphoryla@on sites.
3. Data Analysis: Determines phosphoryla@on paWerns and links them to biological pathways.
2.2. ENRICHMENT OF PHOSPHOPEPTIDES
• Essen@al in phosphoproteomics: phosphorylated proteins and pep@des are present at low abundance and
oQen at low stoichiometry compared to non-phosphorylated forms.
• Without enrichment, the signals from phosphorylated pep@des are typically overshadowed by the more
abundant non-phosphorylated pep@des during mass spectrometry (MS) analysis.
2.2.1. IMMUNO AFFINITY CHROMATOGRAPHY (IAC)
Immuno Affinity Chromatography (IAC)
• Technique to selec@vely capture and isolate specific proteins or pep@des based on their an@genic proper@es
• Using an@bodies (Ab)
An$bodies to capture phosphorylated pep@des
• Ab against pY (phosphotyrosine), pS (phosphoserine), and pT (phosphothreonine) are commonly used
• pS and pT an$bodies: less specific due to poten@al cross-reac@vity with other acidic AA (like Asp and Glu).
• Ab against kinase consensus sequences can also be used for broader specificity.
Applica$ons:
• OQen used in combina$on with other methods like IMAC (immobilized metal affinity chromatography) or
TiO₂ (@tanium dioxide chromatography) to enhance specificity and capture phosphorylated pep@des more
efficiently.
2
, 2.2.2. IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC)
This technique takes advantage of the affinity between nega@vely charged phosphate groups on pep@des and metal
ions immobilized on a sta@onary phase.
Prepara$on of the Sta$onary Phase:
• Metal Chela@on: Metal ions (e.g., Fe³⁺, Ga³⁺) are chelated (chemically bound) to nitrilotriace@c acid (NTA)
ligands, which are pre-aWached to beads.
• This creates a stable metal-NTA complex that forms the sta@onary phase for chromatography.
Binding of Phosphopep$des:
• Phosphopep@des have (-) charged phosphate groups that interact strongly with the (+) charged metal ions.
• The binding is driven by the electrosta@c interac@on between the phosphate groups and the metal centers.
• Non-specific Binding: Other acidic pep@des (rich in aspar@c or glutamic acid residues) or non-phosphorylated
pep@des with anionic features may also bind to some extent, reducing specificity.
Preference for Mul$phosphorylated Pep$des:
• Pep@des with mul@ple phosphate groups exhibit stronger binding due to their higher nega@ve charge and
greater affinity for the sta@onary phase.
Elu$on of Bound Pep$des:
• Elu@on is achieved under basic condi$ons (high pH), which disrupts the interac@on between the phosphate
groups and the metal ions.
2.2.3. VARIATION: SEQUENCTIAL IMAC (SMAC)
3
Voordelen van het kopen van samenvattingen bij Stuvia op een rij:
√ Verzekerd van kwaliteit door reviews
Stuvia-klanten hebben meer dan 700.000 samenvattingen beoordeeld. Zo weet je zeker dat je de beste documenten koopt!
Snel en makkelijk kopen
Je betaalt supersnel en eenmalig met iDeal, Bancontact of creditcard voor de samenvatting. Zonder lidmaatschap.
Focus op de essentie
Samenvattingen worden geschreven voor en door anderen. Daarom zijn de samenvattingen altijd betrouwbaar en actueel. Zo kom je snel tot de kern!
Veelgestelde vragen
Wat krijg ik als ik dit document koop?
Je krijgt een PDF, die direct beschikbaar is na je aankoop. Het gekochte document is altijd, overal en oneindig toegankelijk via je profiel.
Tevredenheidsgarantie: hoe werkt dat?
Onze tevredenheidsgarantie zorgt ervoor dat je altijd een studiedocument vindt dat goed bij je past. Je vult een formulier in en onze klantenservice regelt de rest.
Van wie koop ik deze samenvatting?
Stuvia is een marktplaats, je koop dit document dus niet van ons, maar van verkoper StudentBi0med. Stuvia faciliteert de betaling aan de verkoper.
Zit ik meteen vast aan een abonnement?
Nee, je koopt alleen deze samenvatting voor €14,46. Je zit daarna nergens aan vast.
