New Proteomic Subfractionation Surfaces Innovative Technology For The Improved Resolution of Serum Proteins Swapan Roy, Ph.D., John Krupey, Ph.D., Matthew Kuruc, Devjit Roy ProFACT Proteomics, Inc. Commercialization Center for Innovative Technologies 675 Route One North Brunswick NJ 08902 732-246-1190 (f)732-246-3118 email: mkurucprofactproteomics.com P Pr ro oF FAAC CT T S Su ub bf fr ra ac ct ti io on na at ti io on n P Pr ro oc ce es ss s S Se er ru umm P Pr ro oF FAAC CT T AAn na al ly ys si is s OOp pt ti io on ns s S Su ub bf fr ra ac ct ti io on ns s DDi ir re ec ct tl ly y HHa an nd d- -o of ff f T To o: : Bioassays, eg. Receptor Binding Sugars ELISA Enzyme Protein Interaction Electrophoresis Mass Spec HPLC or CE Carbohydrate Or Phosphorus Bind Washlute E1 ProFACT Proteomics, Inc. Poster Presentation at CHI Biomarker Discovery Summit, September 26,27, 2005 2 ProFACT Proteomics, Inc. Poster Presentation at CHI Biomarker Discovery Summit, September 26,27, 2005 Introduction ProFACT is a new subfractionation methodology designed for comparative proteome analysis. Electrophoretic profiles of serum subfractions demonstrate improved resolution and quantification. Carryover from the three highest abundance serum proteins, Albumin, IgG and Transferrin is minimal. The process starts with a separation platform utilizing a new combination of surface microenvironments substituted with low molecular weight substrates that feature drug- binding motifs. With the ProFACT surface library, undenatured, bioactive proteins can be subfractionated into differential pools. Separations are universal as they do not require pre- qualified binding knowledge, a key limitation of affinity-type techniques. The surfaces utilized are disposable and adaptable to sample size and scale requirements. A simple bind, wash and elute protocol is completed in 30 to 60 minutes and as elutions are mild and consistent, a direct handoff can be made to subsequent interrogation. The interrogation strategy is adaptable to meet investigative inquiry using bottom-up or top-down approaches. Detailed iterative profiling can be applied towards biomarker discovery, disease-state pattern identification, systems biology, or otherwise be useful to reduce sample complexity. The ProFACT surface library can potentially be coupled with HPLC, Capillary Electrophoresis, 1 and 2D Electrophoresis, and Mass Spectrometry to expand coverage and sensitivity. As structural modifications of proteins can alter their binding affinities to the surface library, structural differences in sample sets may be inferred upon interpretation of ProFACT subfraction profiles. The data presented herein demonstrate the unique profiling capabilities of each ProFACT surface library subfraction and the collective resolution of 69 non-redundant proteins, calibrated and quantified from image analysis of SDS-PAGE profiles. 10 differential protein subfractions can be generated in less than 1 hour, without the need for immuno-depletion. Future investigations will focus on comparing normal and disease state sera. Background and Significance Proteomics is encumbered by complexity, unreliable quantification, low-throughput technologies, and lacks systemic ability to uncover structural and functional isoform changes. Recent attention has attempted to reduce the complexity of protein samples, particularly with serum due to the presence of three major protein regions: Albumin, Transferrin and Immunoglobulin. To address this problem, several products have been introduced that have selective binding properties towards one or more of the high abundance proteins in serum. These work through high affinity interactions, most notably using immuno-affinity. Subsequent resolution techniques typically include either 2-dimensional electrophoresis (2DE) or multi-dimensional HPLC. While productive, these methods generally are: ? costly for large sample sets, ? cumbersome and low throughput, ? at best only moderately quantifiable, ? generate limited structural information, ? not providing intact, bioactive protein pools. ProFACT is a new subfractionation surface library intended to address these fundamental problems. 3 ProFACT Proteomics, Inc. Poster Presentation at CHI Biomarker Discovery Summit, September 26,27, 2005 Complexity Reduction Strategy The ProFACT process begins by first subfractionating serum, then analyzing, comparing and contrasting each subfraction individually. 1-dimensional electrophoresis is well-suited for this task as it is quick, reproducible and offers precise peak resolution capability and relative peak calibration and quantification after analysis through commercially available image software