Direct Selection of Monoclonal Phosphospecific Antibodies without Prior Phosphoamino Acid Mapping

Abstract

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 upfront 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 animalbased 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, nonprofit 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, co...

Reconstitution of the cell cycle-regulated Golgi disassembly and reassembly in a cell-free system

“…Then directly fix the reaction and analyze by EM (Steps 60–73). Alternatively, analyze disassembly and reassembly reactions by western blotting for phosphorylation of Golgi structural proteins 10,21,23,24,41,47 .…”

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

“…Although phosphospecific antibodies have been obtained successfully from classical naive antibody fragment libraries (35), it is often the case that functional application of such antibodies to therapeutic or diagnostic settings requires a process of in vitro affinity maturation, which can be technically challenging, time consuming, and costly (59,60). Additionally, if they are generated against full-length phosphorylated protein, then the epitopes recognized by the antibodies cannot be dictated and must be elucidated post hoc (35).…”

“…Additionally, if they are generated against full-length phosphorylated protein, then the epitopes recognized by the antibodies cannot be dictated and must be elucidated post hoc (35).…”

“…Although the recognition of phosphopeptides by protein kinases and phosphatases has been extensively studied (33), there are only a handful of papers describing anti-phosphopeptide antibodies that were selected via display technologies (34,35). Furthermore, none of the phosphopeptide-antibody complexes currently described in the literature have been structurally characterized beyond in silico modeling and CDR mutagenesis (36 -38).…”

Aβ-induced Golgi fragmentation in Alzheimer’s disease enhances Aβ production

“…For kinase inhibition, cells were treated with roscovitine for an additional 2 h in the presence of Aβ42. For TAT-CIP treatment, cells were pretreated with 600 nM TAT-CIP or TAT-GFP for 30 min, Aβ42 was added, and the cells were incubated for another 12 h. Cells were lysed and evaluated by Western blot analysis using antibodies for phospho-GRASP65 (LX108) or total GRASP65 (Mary) (32,48).…”