cleavage sites and binding affinities
DESCRIPTION
Cleavage sites and binding affinities. Cleavage measurements. Peptide Digest Workflow. Purification of Peptides by RP-HPLC: Synthesized peptides are polished to >98% purity (1h Gradient, 25 cm x 4 mm C18 column), lyophilized and used for digestion - PowerPoint PPT PresentationTRANSCRIPT
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Cleavage sites and binding affinities
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Cleavage measurements
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Peptide Digest Workflow
Purification of Peptides by RP-HPLC:Synthesized peptides are polished to >98% purity (1h
Gradient, 25 cm x 4 mm C18 column), lyophilized and used for digestion
Digestion: 100 ul Volume - 15 ul/ TimepointMSMS-Analysisvery fast – analysis of peptide digests can be performed in one
day multiple time points possible (Instrument time: 90 min/timepoint)Data Processing:Waters Protein Expression System (1-2h processing)Excel-Macros (Manual, 30 min/timepoint)
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BiC BioCentrum-DTUTechnical University of Denmark
Peptide polishing:
very high purity of peptide substrates required, but some peptides ordered at >80% purity
are quite „dirty“ -> time consuming „polishing“ of peptides (4-8h)
Protocol has been established for routine purification
high hydrophobicity of some peptides leads to solubility /purification problems
Different/modified selection criteria for next QBC set?
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BiC BioCentrum-DTUTechnical University of Denmark
Expression Data Acquisition
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MHC Binding
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BiC BioCentrum-DTUTechnical University of Denmark
RMAS Assay: classical way to measure peptide binding- However not quantitative (no determination of the affinity)
TAP difficient cell line
At 37 °C At 26 °C
Add peptide
Measure T cell activation
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Experimental description of peptide-MHC binding
Hans-GeorgRammensee et al.,
www.syfpeithi.de
Søren Buus et al.,
www.cbs.dtu.dk/services/NetMHC/
How to examine HLA specificity?
”What the HLA has bound in vivo” Elution and sequencing of natural ligands
Simpel motif ~ low sensitivity predictions
”What the HLA will, or will not, bind in vitro” Determine the binding strength of any peptide
Extended motif ~ higher sensitivity predictions
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How to determine peptide affinityHow to determine peptide affinityLaw of mass action
[R] + [L] [RL]
koff
kon
KD = koff (S-1)/kon (M-1S-1)
Peptide [M]
Bin
ding
KD = (10-15-10-6 M)
100%
50%
Peptide Log [M] Cold Peptide Log [M]
Log IC50B
ind i
n g h
o t p
eptid
e
Bi n
ding
[MHC] + [P] [P*MHC]
koff
kon
Saturation assay
Inhibition assay
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BiC BioCentrum-DTUTechnical University of Denmark
How to do radioactive biochemical inhibition binding assays
• Obtain purified HLA
• Or recombinant heavy chain & b2m
• Obtain indicator peptide
• Perform dose titration of any inhibitory peptide
• Separate free from bound peptide
• Calculate binding and IC50
Non binding test peptide
Binding test Peptide
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BiC BioCentrum-DTUTechnical University of Denmark
A spun column binding assayA spun column binding assay
G50
b2m
MHC
peptide
Binding test peptid
Non binding test peptid
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PROSPROS
• Truly quantitativeTruly quantitative
• Can address affinities in Can address affinities in
the low nM levelthe low nM level
• ReproducibleReproducible
CONS
• Radioactive
• Not a standard method
• Waste problem
The radioactive biochemical binding assay
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BiC BioCentrum-DTUTechnical University of Denmark
The Quantitative ELISA Capable of Determining Peptide-MHC Class I Interaction
Made possible by our recent development of highly active recombinant MHC class I heavy chains– functional equivalents of ”empty” molecules
Pros:• Reasonably simple, sensitive and quantitative• Does not depend on labeled peptide• It is easily adaptable to other laboratories
Disseminated protocol and standard reagents
L.O.Pedersen et al., , EJI. 2001, 31: 2986
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DevelopmentDevelopment
Sensitivity below 0.1 nM or 5 x 10Sensitivity below 0.1 nM or 5 x 10-15-15 M M MHC class I complex !MHC class I complex !
Strategy for the assay
Step II: Detection of de novo folded MHC class I molecules by ELISA
IncubationIncubation
Step I: Folding of MHC class I molecules in solution
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BiC BioCentrum-DTUTechnical University of Denmark
nM c
ompl
ex d
etec
ted
nM peptide offered
Results are expressed as:BMAX : Amount of detected complex
including 95% confidence interval
ELISA driven assay
KD: Peptide affinityincluding 95% confidence interval
Concentrations of complexes generated are plotted as a function of the concentration of peptide offered
Sylvester-Hvid, C. et al., Tissue Antigens (2002) 59:251
Automated, 384 format
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BiC BioCentrum-DTUTechnical University of Denmark
Pros– Homologous proximity assay.– No washing => fast development
AlphaScreen
Acceptor bead
Donor bead
<200 nmO2•
Cons– Expensive reagents– Specialized reader
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BiC BioCentrum-DTUTechnical University of Denmark
560 nM
480 nM
Biotin
Streptavidin
anti-mouse IgG
W6/32 mouse anti-HLA
AlphaScreen
O2•
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BiC BioCentrum-DTUTechnical University of Denmark
AlphaScreen
0
1
10
100
1.000
10.000
100.000
0 1 10 100 1.000 10.000 100.000
ELISA KD nM
Alp
haS
cree
n K
D n
M
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BiC BioCentrum-DTUTechnical University of Denmark
TAP Binding
• TAP is very hard to purify for in vitro assays
• Most used assay is the radioactive RIA assay
• IC50 from RIA assays are dependent of the affinity of the competing peptide