1. FUNCTIONALIZED COLLOIDAL NANOPARTICLES Jaan Noolandi

2. BACKGROUND Proteome – PROTEins expressed by a genOME Study behavior of system to produce a living organism Complexity requires new analytical tools for greater versatility and higher throughput than existing methods Emphasis on multiplexing and miniaturization Current Methods Protein microarrays (chips) Self-encoded latex beads Submicrometer metallic barcodes Functionalized nanoparticles

3. Protein mixture Proteins PeptidesMS analysis MS data Database search algorithms identification digestion Protein mixture digestion separation General flow scheme for proteomic analysis “Introduction to Proteomics - Tools for the New Biology” Daniel C. Liebler, Humana Press (2002)

4. “Mass Spectrometry and Proteomics” Steve P. Gygi and Ruedi Aebersold Current Opinion in Chemical Biology 4, 489-494 (2000)

5. “Protein Arrays” New Focus, Science 289, 1673 (2000)

6. “Optical Coding of Biomolecules” Shuming Nie et al Nature Biotechnology 19, 631-635 (2001)

7. “Submicrometer Metallic Barcodes” Christine D. Keating, Michael J. Natan et al Science 294, 137-141 (2001) Synthesis of barcoded particles.

9. Synthesis of Functional Colloidal Nanoparticles  Monodisperse arm lengths to obtain well-defined mobility of “bare” particle  Optimize arm lengths  Optimize arm density

11. Capillary Electrophoresis

12. Bioaffinity Capillary Electrochromatography

13. “Bait” approach to detection of DNA-interacting protein with a functionalized colloidal particle probe. protein extract Functionalized colloidal particle probe separate by capillary electrophoresis Identify Digest, MS analysis Probe-protein complex

14. SYNTHESIS STRATEGY Hydrophobic / hydrophilic block copolymer - hydrophilic block (sulfonated polystyrene, maleic anhydride) - hydrophobic block (polystyrene) Micelle formation - add monomer, initiator - form miniemulsion - polymerize Photo-emulsion polymerization - core latex particles by emulsion polymerization - add polymerizable photoinitiator - polymer brush by emulsion polymerization

15. end functionalize water-soluble polymer with maleimide group Conjugation of Biomolecules with Synthetic Polymers Reaction of a maleimide-terminated PEG polymer with a 5’-thiol-terminated ssDNA oligomer O O N N SH S O O O O FAM-(dT) 20 or 35 NH(CH 2 -CH 2 O) n H

16. SUMMARY Increase throughput of proteomics assays by combining capillary electrophoresis involving colloidal particles characterized by well-defined molar mass profile with massive parallel processing by capillary arrays Colloidal particles synthesized by different synthetic routes (photo-emulsion polymerization, micelle formation, living radical polymerization)