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Comprehensive Peptide Cleavage and MHC Binding Affinity Analysis Workflow

This document provides a detailed workflow for peptide purification, cleavage measurements, and MHC binding affinity analysis. It covers RP-HPLC purification methods achieving >98% purity, highlights the rapid MSMS analysis of peptide digests in just one day, and describes various assays such as classical measurement techniques and quantitative ELISA protocols. Key considerations include purification challenges related to hydrophobicity, binding strength assessment using the law of mass action, and innovative methodologies for sensitive detection of peptide-MHC class I interactions.

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Comprehensive Peptide Cleavage and MHC Binding Affinity Analysis Workflow

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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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