TY - JOUR
T1 - Immuno-proteomics
T2 - Development of a novel reagent for separating antibodies from their target proteins
AU - Ganesan, Vinitha
AU - Schmidt, Brigitte
AU - Avula, Raghunandan
AU - Cooke, Dagney
AU - Maggiacomo, Taylor
AU - Tellin, Lawton
AU - Ascherman, Dana P.
AU - Bruchez, Marcel P.
AU - Minden, Jonathan
N1 - Funding Information:
We would like to thank Malachi Blundon, Anna Grace Pyzel, Danielle Schlesinger and Dr. Fred Lanni for helpful discussions on this work. We thank Dr. Tina H. Lee for allowing us to use their cell culture facility. B.S. and M.P.B. were supported by NIH TCNP grant U54GM103529 . R.A. D.C. T.M. and L.T. were supported by an HHMI (grant 52006917 ) Undergraduate Science Education award.
Publisher Copyright:
© 2014 Elsevier B.V.
PY - 2015/6/1
Y1 - 2015/6/1
N2 - Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics.
AB - Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics.
KW - Immuno-proteomics Serological proteomics Difference gel electrophoresis (DIGE) Reversible biotin Immunoprecipitation
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U2 - 10.1016/j.bbapap.2014.10.011
DO - 10.1016/j.bbapap.2014.10.011
M3 - Article
C2 - 25466873
AN - SCOPUS:84961327949
VL - 1854
SP - 592
EP - 600
JO - BBA - Protein Structure
JF - BBA - Protein Structure
SN - 1570-9639
IS - 6
ER -