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Cleavage sites and binding affinities

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

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Cleavage sites and binding affinities

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  1. Cleavage sites and binding affinities

  2. Cleavage measurements

  3. 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/ Timepoint • MSMS-Analysis • very 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)

  4. 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?

  5. Expression Data Acquisition

  6. MHC Binding

  7. Measure T cell activation RMAS Assay: classical way to measure peptide binding - However not quantitative (no determination of the affinity) At 26 °C At 37 °C Add peptide TAP difficient cell line

  8. Hans-Georg Rammensee et al., www.syfpeithi.de Søren Buus et al., www.cbs.dtu.dk/services/NetMHC/ Experimental description of peptide-MHC binding 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

  9. Peptide Log [M] KD = (10-15-10-6 M) How to determine peptide affinity Law of mass action koff koff [R] + [L] [RL] [MHC] + [P] [P*MHC] kon kon KD = koff (S-1)/kon (M-1S-1) Saturation assay Inhibition assay 100% Binding Binding hot peptide 50% Binding Peptide [M] Cold Peptide Log [M] Log IC50

  10. Binding test Peptide Non binding test peptide 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

  11. Non binding test peptid Binding test peptid A spun column binding assay MHC b2m peptide G50

  12. The radioactive biochemical binding assay • CONS • Radioactive • Not a standard method • Waste problem • PROS • Truly quantitative • Can address affinities in the low nM level • Reproducible

  13. 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 L.O.Pedersen et al., , EJI. 2001, 31: 2986 • Pros: • Reasonably simple, sensitive and quantitative • Does not depend on labeled peptide • It is easily adaptable to other laboratories • Disseminated protocol and standard reagents

  14. Incubation Development Sensitivity below 0.1 nM or 5 x 10-15 M MHC class I complex ! Strategy for the assay • Step II: Detection of de novo folded MHC class I molecules by ELISA • Step I: Folding of MHC class I molecules in solution

  15. ELISA driven assay Results are expressed as: BMAX : Amount of detected complex including 95% confidence interval KD: Peptide affinity including 95% confidence interval nM complex detected Automated, 384 format nM peptide offered 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

  16. AlphaScreen • Pros • Homologous proximity assay. • No washing => fast development • Cons • Expensive reagents • Specialized reader Donor bead Acceptor bead O2• <200 nm

  17. O2• AlphaScreen 560 nM 480 nM Biotin Streptavidin anti-mouse IgG W6/32 mouse anti-HLA

  18. AlphaScreen

  19. 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

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