Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Abstract

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Much protein activity is regulated by post-translational modifications (PTMs), 1 over 200 of which have been described (1), with changes in enzymatic activity, protein interaction partners, subcellular localization, and targeted degradation being some of the most important regulated activities. A number of different approaches have been used to study PTMs, most of which rely on either specific isolation of the protein of interest and identification of the PTMs that affect that particular protein (2) or isolation of all proteins containing a specific PTM of interest and subsequent identification of the isolated proteins. There are two basic methods to isolate or identify all proteins containing a specific PTM: chemical derivatization or specific affinity reagents. The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5-8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17). Although antibodies are by far the most common specific affinity reagents, there are very few that recognize PTMs independently of the proteins to which they are attached. Nitrosylated tyrosine and phosphorylated tyrosine are two exceptions for which antibodies have been used in proteomics studies (13-17), and antibodies against phosphoserine/threonine have also been identified (18), indicating that the utility of such reagents...

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

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Toward a framework for sulfoproteomics: Synthesis and characterization of sulfotyrosine‐containing peptides

“…127 While it has been rather straightforward to obtain antibodies that recognize specific sulfoTyr-containing epitopes, 123 attempts to derive anti-sulfoTyr antibodies that recognize sulfoTyr independently of the sequence context by immunization methods have failed. 123,128 This lack of an immune response against sulfoTyr might be explained by the presence of the modification in a large number of secreted and transmembrane proteins, rendering sulfoTyr non immunogenic by itself. However, more recently sequence-independent anti-sulfoTyr monoclonal antibodies have been obtained by an alternative approach to immunization methods using phage display technology which circumvents the innate tolerance of the immune system toward this modification.…”

“…However, more recently sequence-independent anti-sulfoTyr monoclonal antibodies have been obtained by an alternative approach to immunization methods using phage display technology which circumvents the innate tolerance of the immune system toward this modification. 61,128 The antibodies were shown to be specific for sulfoTyr and did not recognize Tyr, phosphoTyr or sulfated glycans. Furthermore, Hoffhinen et al also demonstrated that their antibody could be used for the selective enrichment of sulfoTyr-containing proteins from complex biological samples for further analysis by MS. …”

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“…Since the sulfate moiety at the tyrosines is chemically unstable, we confirmed the integrity of the synthesized peptides by their reactivity with the sulfotyrosine-specific antibody 1C-A12 ( Fig. 2A, bottom panel) (30). Binding of R5, X4, or dualtropic Env to the peptide membranes was analyzed both after preincubation with sCD4 and with no preincubation with sCD4 (Fig.…”