%0 Journal Article
%T Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform
%A Amita R. Oka
%A Matthew P. Kuruc
%A Ketan M. Gujarathi
%A Swapan Roy
%J International Journal of Proteomics
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/515372
%X Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling. 1. Introduction Proteomic profiling based on enzyme activity is assuming significance in drug discovery as it becomes possible to profile selectivity of drugs and their mechanism of action [1]. Such an approach focuses on protein function, an aspect which has been missing from expression proteomics [1]. A functional proteomic profiling approach has the potential not only to help identify targets for diagnosis and therapy [2], specifically in personal medicine [3, 4], but also to reveal the underlying mechanisms of action of disease-sustaining proteins [5]. Methods for global analysis of protein expression and function, including liquid chromatography with mass spectrometry (MS) for shotgun analysis [6, 7], yeast two-hybrid methods [8], and protein microarrays [9], have been crucial in developing the field of proteomics, but they do not provide an accurate assessment of functional states of proteins in cells and tissues [10]. Activity-based protein profiling (ABPP) was first demonstrated for serine hydrolyses [11] and has now been applied to other enzyme classes such as kinases, phosphatases, and
%U http://www.hindawi.com/journals/ijpro/2012/515372/