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Phospho-specific antibodies by design
Oda Stoevesandt & Michael J Taussig
Antibodies specific to phosphorylated peptides are readily generated through structure-based engineering and in vitro selection.
npg 2013 Nature America, Inc. All rights reserved.
The study of post-translational modification of proteins, a critically important aspect of cell physiology and pathology, depends on the availability of specific antibody reagents. In many cases, however, it is difficult to generate these reagents because of the difficulty of focusing the antibody response appropriately in conventional immunization of animals. In this issue, Koerber et al.1 present what could be a game-changing strategy for producing recombinant antibodies that recognize the most widely studied post-translational modification, phosphorylation, with possible extension to other types of modification. Their structurally informed approach, coupled with in vitro phage display selection, opens up the possibility of producing a wider range of affinity probes for the huge diversity of modified proteins in proteomes.
Reversible phosphorylation of many cellular proteins regulates their activity in a number of ways, including switching on or off protein interactions in signal transduction pathways, changing the function of transcription factors or altering their subcellular localization. The principal phosphorylated amino acid residues are phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr), the last of these being the best studied, particularly for its key role in signal transduction2. Uncontrolled phosphorylation through aberrant protein kinase activation is responsible for many cancerous changes and is a major target of therapeutic interventions. Accurate detection of protein phosphorylation and other post-translational modification is therefore central to understanding protein function and to monitoring the state of the cell.
Mass spectrometry is the gold standard for large-scale identification and quantification of phosphoproteins3, but antibodies are indispensable for their detection in enzyme-linked immunosorbent assays (ELISA), western blot analysis, immunoimaging, proximity ligation, protein microarrays and flow cytometry, and for affinity purification. Reagents are available that bind phosphorylated amino-acid residues regardless of protein sequence, but producing antibodies that recognize the modification in the context of its individual protein sequence is more challenging4. Typically, this involves immunizing animals with phosphopeptides to prepare either polyclonal antibodies, rendered specific through extensive purification, or monoclonal antibodies, by screening large numbers of candidate hybridoma clones against phosphorylated and nonphosphorylated targets.