slide presentation of multiplexed high throughput screening assay for protein - protein interactions and applications of this approach

1. Proteome screening for macromolecular complexes in molecular medicine Candace Strang PhD 2009

2. General theme DETECTprotein binding pair- Analytical detection by high throughput screen Conditions can be varied simply, cheaply CONFIRMmolecular interaction at the structural level- Biochemical characterization by IP / MS ESTABLISH biological significance- Confirmed by the identification of intracellular interactions that are crosslinked in vivo, then characterized as above

3. Part I DETECTprotein binding pair- Analytical detection by high throughput screen Conditions can be varied simply, cheaply

4. proof of concept Schematic of C1 binding to an immune complex C1=C1q+2C1s+2C1r C1q C1r2C1s2 C1 IgG

5. mid-complex location for C1r, C1s EM of C1q v. C1 reveals coronal viewsagittal view C1q C1q C1 C1

6. Suggestion– there is a modification to a well-established Q-ELISA format, that would allow for a screen for interacting proteins. First, recall a Quantitative ELISA format - Trap protein of interest with a monoclonal AB, allow- ingthe protein to be uniformly oriented and mostly free Bathe the mAB with your physiological fluid or culture isolate of choice Detect protein of interest with a polyclonal AB, allowing the protein to be bound with multiple AB molecules to optimize the signal from biotin Detect the protein of interest with sensitive laser and ~irreversible formation of signal

7. Modification for the detection of protein complexes Suggestion– using this same Q-ELISA based format, with the monoclonal capture antibody, and a 2nd detection antibody, one can change the detection antibody to an AB (Layer 3) that recognizes the interaction partner to identify the macromolecular complex, and characterize the conditions under which the complex is present or absent.

8. Q-ELISA technique – current uses Uses standard clinical assay Q-ELISA based instrumentation, developed for high throughput, minimal sample volume, has been verified by numerous assays for several antigens, by commercial kit or customized design Kits already in use Apoptosis Signal transduction, AKT pathway Acute phase response panel Inflammation panel Death receptor pathway Growth factors Lipoprotein panel, cardiac evaluation

10. pAB –biotin for the detection step

12. Run the assay using the standardized control apolipoprotein to confirm that the reagents are working as expected.

14. Examples for the components of C1 and the C1 complex of 3 proteins Single component detection *Quantitative difference likely due to Change in C1q level due to loss associated with blood clotting and clot removal from the serum, or an increase in accessibility of the C1q epitopes when the protein is free (plasma) v. in complex, (serum), since C1s, C1r coat the 6 arms of the C1q molecule. Quantization of C1s is similar for free v. complexed protein C1s is free in plasma, in the C1 complex in serum ***Since the C1s concentration does not change appreciably between serum and plasma, it is likely that (2) is the reason for the change in C1q concentration.***

15. Examples for the components of C1 and the C1 complex of 3 proteins Q S R Q S R Q QQ C1q C1 C1 Capture / trapping Detection Information Yield

16. Examples for the components of C1 and the C1 complex of 3 proteins Q S R Q S R S SS C1 C1s C1r2C1s2 Capture /trapping Detection Information Yield

17. Part II CONFIRMmolecular interaction at the structural level- Biochemical characterization by IP / MS

18. Biochemical proteomics Further molecular analysis at the biochemical level- magnetic beads to capture the immune complex, followed by further screening, including MS AB-AG

19. Part III ESTABLISH biological significance- Confirmed by the identification of intracellular interactions that are crosslinked in vivo, then characterized as above, Part I, II

20. Intracellular ID of complex, using biosynthetically labeled proteins with highly reactive and nonspecific AA chemistry, either diazirine- M, or L, or acetylene-M.

21. Applications Cell signalling biology Protein – protein interactions that change to initiate, or, as a result of, signal transduction -using physiological conditions, screen for signal - specific interactions- such as ligand binding, metal binding, oxidative stress, Calcium concentration, low Oxygen tension, other pathway signals, that will change on signal transduction, or with the onset of pathophysiology. Document the correlation between signal and the protein – protein interaction in question.

22. Applications 2 Factor assessment for complex formation Determine the physical factors that allow optimal complex formation, rapidly, with a minimal amount of material, and under a range of conditions.

23. Applications 3 Quality control Screen recombinant proteins v. endogenous, native proteins. Determine if an antibody that is generated to a peptide, can then recognize the folded domain or protein. Screen proteins that have been altered by engineering, mutation, or chemical changes Optimize conditions to study the interaction of interest.

24. Applications 4 General screen for interaction partners It is possible to use a tag antibody, such as anti-His, to produce a general screen for protein – protein interactions where one partner is not known. Furthermore, it is possible to use an antibody that targets a specific chemical modification, such as biotin, thereby allowing for screening of a mixture of unknown proteins for either the capture or detection sample.

25. Technical details Part I Assay format (96 well plate, complex formation on beads, streptavidin-biotin detection, laser detection, single bead analysis) is well established. Adequate technical support is available. Assay design has been validated on a complex that is well understood, from both a physiologic standpoint, and a biophysical standpoint. Assay format is suitable for complex mixtures, in a physiologic background. Assay design is viable for proteins that are minor components of the protein milieu. Part II This aspect of the program is in place to allow biochemical and biophysical confirmation of complex formation. It capitalizes on the optimization screening that is carried out in Part I, by using a similar format (immunocapture on beads), but in the larger scales required for biochemical and biophysical analysis, in comparison to laser based fluorescence detection. Part III Familiarity with the nuances of crosslink formation in proteins is in place from previous work. Cell biology is in place from previous work. Assays from Part I can be used to screen for crosslink formation, again requiring small amounts of material for reliable screening, to compensate for possible low yields @ the crosslink and harvesting steps. Methods do not require radioactivity.

26. Acknowledgements Solomon Murphy, chief cook and bead washer Seok J Hong, Gregory M Landes, Moral support Arthur Sands, CEO, Lexicon Pharmaceuticals