Producing peptide arrays for epitope mapping by intein-mediated protein ligation
Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations,...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2004-09-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/04373RR01 |
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| author | Luo Sun John Rush Inca Ghosh Jeremy R. Maunus Ming-Qun Xu |
| author_facet | Luo Sun John Rush Inca Ghosh Jeremy R. Maunus Ming-Qun Xu |
| author_sort | Luo Sun |
| collection | DOAJ |
| description | Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 104-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications. |
| format | Article |
| id | doaj-art-63a5416e9d9b451db09b4524f58f5aac |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2004-09-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-63a5416e9d9b451db09b4524f58f5aac2025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182004-09-0137343044310.2144/04373RR01Producing peptide arrays for epitope mapping by intein-mediated protein ligationLuo Sun0John Rush1Inca Ghosh2Jeremy R. Maunus3Ming-Qun Xu41New England Biolabs, Beverly2Cell Signaling Technology, Beverly, MA, USA1New England Biolabs, Beverly1New England Biolabs, Beverly1New England Biolabs, BeverlyPeptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 104-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications.https://www.future-science.com/doi/10.2144/04373RR01 |
| spellingShingle | Luo Sun John Rush Inca Ghosh Jeremy R. Maunus Ming-Qun Xu Producing peptide arrays for epitope mapping by intein-mediated protein ligation BioTechniques |
| title | Producing peptide arrays for epitope mapping by intein-mediated protein ligation |
| title_full | Producing peptide arrays for epitope mapping by intein-mediated protein ligation |
| title_fullStr | Producing peptide arrays for epitope mapping by intein-mediated protein ligation |
| title_full_unstemmed | Producing peptide arrays for epitope mapping by intein-mediated protein ligation |
| title_short | Producing peptide arrays for epitope mapping by intein-mediated protein ligation |
| title_sort | producing peptide arrays for epitope mapping by intein mediated protein ligation |
| url | https://www.future-science.com/doi/10.2144/04373RR01 |
| work_keys_str_mv | AT luosun producingpeptidearraysforepitopemappingbyinteinmediatedproteinligation AT johnrush producingpeptidearraysforepitopemappingbyinteinmediatedproteinligation AT incaghosh producingpeptidearraysforepitopemappingbyinteinmediatedproteinligation AT jeremyrmaunus producingpeptidearraysforepitopemappingbyinteinmediatedproteinligation AT mingqunxu producingpeptidearraysforepitopemappingbyinteinmediatedproteinligation |