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NOVEL METHODS FOR TARGET PROTEIN IDENTIFICATION USING IMMUNOPRECIPITATION - LC/MS/MS
Citation:
Winnik, W. M., R D. Grindstaff, L B. Copeland, AND MDW Ward. NOVEL METHODS FOR TARGET PROTEIN IDENTIFICATION USING IMMUNOPRECIPITATION - LC/MS/MS. Presented at Genetics and Environmental Mutagenesis Society, Chapel Hill, NC, November 10, 2004.
Description:
Proteomics provides a powerful approach to screen and analyze responses to environmental exposures which induce alterations in protein expression, phosphorylation. ubiquitinylation, oxidation. and modulation of general proteome function. Post-translational modifications (PTM) of proteins have been shown to be associated with key events in tumorgenesis, altering cell cycle progression, apoptosis induction, cellular differentiation, and DNA repair.
In these studies, we focus on identifying specific subsets of the proteome using immunopurification of targeted proteins. After immunoprecipitation, all proteins were size-separated by SDS-PAGE, then followed by in-gel tryptic digestion. Finally, peptides could be identified by nano-flow LC/MS/MS.
By immunoprecipitating proteins that are ubiquitinylated and/or phosphorylated we could focus on posttranslational modifications that may be related to a biomarker of chemical exposure and/or disease state. To identify PTM of proteins, we first coupled anti-ubiquitin, anti-phosphotyrosine and anti-phosphothreonine to protein G coated Sepharose beads, then incubated control or chemically treated whole cell lysates with the immune complex
Also, we are developing a method for the purification and subsequent characterization of IgE inducing proteins in mold extracts (Metarhizium anisopliae mycelium) by immunoprecipitating allergen specific immunoglobulins. Briefly, by covalently linking an anti-IgE antibody to chemically modified magnetic beads, we were then able to pull down the IgE from mouse serum derived from M anisopliae immunized mice. This anti-IgE - IgE complex was used to immunoprecipitate the proteins in mold extracts that induce an IgE immune response. In conclusion, by immunopurifying target proteins from a complex mixture we selected a precise subset of the proteome, resulting in less complex improved protein identification and analysis.