Direct Selection of Monoclonal Phosphospecific Antibodies without Prior Phosphoamino Acid Mapping

In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.In the current post-genomic era, large scale efforts are underway to study the emerging proteome. Because of their ability to bind their respective targets with extremely high specificity, antibodies are essential tools in this endeavor. In fact, non-profit organizations like the European “ProteomeBinders” Consortium (1) and the “Clinical Proteomic Technologies for Cancer” network (2) as well as commercial enterprises are currently sponsoring large projects to assemble all-inclusive antibody libraries.To regulate their function, many proteins are altered after their initial synthesis. Because such post-translational modifications change the physicochemical properties of a given protein, this further increases the complexity of the proteome. Therefore, the true challenge is to have not only all-inclusive antibody libraries but also to assemble collections of sensors, which specifically detect post-translational changes.One of the best studied modifications after polypeptide synthesis is reversible protein phosphorylation through the action of specific kinases and phosphatases. A plethora of cellular processes hinges upon the correct phosphorylation state of key regulators (3), and errors may result in cell death or malignancy (4). It is therefore not surprising that protein phosphorylation represents a very active area of research in the quest for new therapeutic cancer targets (5). Phosphospecific antibodies, first described over 25 years ago (6–8), represent key tools in the study of cellular processes regulated by phosphorylation. Their production, however, is relatively costly and time consuming.In our laboratory we have succeeded in obtaining highly functional, conformation-specific recombinant antibodies using the method of phage display (9, 10).⁵ This not only allowed us to follow the fate of proteins upon activation but also demonstrated that the diversity of available recombinant antibody libraries is sufficiently broad. We thus decided to use a similar approach for the selection of phosphospecific antibodies. We chose GRASP65 as the antigen, the predominant phosphoprotein of mitotic Golgi membranes (11). Careful studies have mapped several phospho-residues of this structural Golgi protein, and phosphospecific antibodies have been generated using classical methods (12–14). This study describes a novel approach, which allowed the direct selection and characterization of three distinct and highly functional monoclonal phosphospecific antibodies.