1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Cleavage sites and binding affinities

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Cleavage measurements

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical 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?

5. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Expression Data Acquisition

6. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark MHC Binding

7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical 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

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Experimental description of peptide-MHC binding Hans-Georg Rammensee 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

9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark How to determine peptide affinity Law of mass action [R] + [L] [RL] k off k on K D = k off (S -1 )/k on (M -1 S -1 ) Peptide [M] Binding K D = (10 -15 -10 -6 M) 100% 50% Peptide Log [M] Cold Peptide Log [M] Log IC 50 Binding hot peptide Binding [MHC] + [P] [P*MHC] k off k on Saturation assay Inhibition assay

10. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical 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 IC 50 Non binding test peptide Binding test Peptide

11. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark A spun column binding assay G50 b2m MHC peptide Binding test peptid Non binding test peptid

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark PROS Truly quantitative Truly quantitative Can address affinities in the low nM level Can address affinities in the low nM level Reproducible Reproducible CONS Radioactive Not a standard method Waste problem The radioactive biochemical binding assay

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical 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

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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 Incubation Step I: Folding of MHC class I molecules in solution

15. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark nM complex detected nM peptide offered Results are expressed as: B MAX : Amount of detected complex including 95% confidence interval ELISA driven assay K D : Peptide affinity including 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

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Pros –Homologous proximity assay. –No washing => fast development AlphaScreen Acceptor bead Donor bead <200 nmO 2 Cons –Expensive reagents –Specialized reader

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 560 nM 480 nM Biotin Streptavidin anti-mouse IgG W6/32 mouse anti- HLA AlphaScreen O 2

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark AlphaScreen

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark TAP Binding TAP is very hard to purify for in vitro assays Most used assay is the radioactive RIA assay IC 50 from RIA assays are dependent of the affinity of the competing peptide