Subject(s)
Cell Line, Tumor , Drug Screening Assays, Antitumor , Melanoma/metabolism , Antigens, Neoplasm/metabolism , Cell Differentiation , Cluster Analysis , Female , Gene Expression Regulation, Neoplastic , HLA Antigens/metabolism , Humans , Male , Mutation , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins B-raf/metabolism , ras Proteins/metabolismABSTRACT
Immobilizing chemically synthesized analogues of PI(3,4,5)P3 onto Affi-10 beads and incorporating them into liposomes allowed their use as affinity absorbents in the comprehensive analysis of the phosphoinositide interactome using cytosolic cell extracts of the LIM1215 colon cancer cell line. This led to the identification of 282 proteins that either interact with PI(3,4,5)P3 or are indirectly captured as part of a complex containing a PI(3,4,5)P3 binding partner. Identification of the proteins was achieved using affinity/LC-MS/MS experiments.
Subject(s)
Carcinoma/metabolism , Colonic Neoplasms/metabolism , Computational Biology/methods , Phosphatidylinositol Phosphates/chemistry , Proteomics/methods , Cell Line, Tumor , Cytosol/metabolism , Glutathione Transferase/metabolism , Humans , Liposomes/chemistry , Mass Spectrometry/methods , Models, Chemical , Phosphates/chemistry , Phosphatidylinositol Phosphates/metabolism , Protein Interaction Mapping , ProteomeABSTRACT
A comprehensive analysis of the phosphoinositide interactome has been performed using analogues of PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids which were immobilized onto Affi-10 beads or incorporated into liposomes for use as affinity absorbents with cytosolic extracts from colonic carcinoma cell lines. Affinity/LC/MS/MS experiments allowed identification of 388 proteins/protein complexes that appeared to interact specifically with the phosphoinositide targets: a number of novel potential phosphoinositide interacting proteins have been identified.
Subject(s)
Gene Expression Regulation, Enzymologic , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol Phosphates/chemistry , Proteomics/methods , Cell Line, Tumor , Chromatography, Ion Exchange/methods , Cytosol/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Liposomes/chemistry , Magnetic Resonance Spectroscopy , Peptides/chemistry , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Phospholipids/chemistry , Phosphorylation , ProteomeABSTRACT
A small series of derivatives of the alkaloid naamidine A was synthesized and tested in vitro for their ability to inhibit mitogenesis in BaF/ERX cells. Replacement of the imidazole core with a thiazole was found to have only a minor effect on potency, and the 4-methoxybenzyl substituent of the natural product was shown to be unnecessary for activity.
Subject(s)
Antineoplastic Agents/pharmacology , Chemistry, Pharmaceutical/methods , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Animals , Cell Line , Drug Design , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Imidazoles/chemistry , Inhibitory Concentration 50 , Mice , Models, Chemical , Porifera , Structure-Activity Relationship , Thiazoles/chemistryABSTRACT
Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.
