Subproteomics analysis of phosphorylated proteins: Application to the study of B-lymphoblasts from a patient with Scott syndrome

Proteomics based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly used to address biomedical questions. Proteins are the main functional output, and post‐translational modifications such as phosphorylation are very important in determining protein function. To address this question, we developed a method for specific immunoprecipitation using anti‐phosphotyrosine antibodies. This method is directly compatible with two‐dimensional gel electrophoresis (2‐DE). In this report data are presented on B‐lymphoblasts from a patient suffering of Scott syndrome. Scott syndrome is an orphan inherited hemorrhagic disorder due to a lack of exposure of procoagulant phosphatidylserine at the exoplasmic leaflet of plasma membrane of blood cells. We hypothesized that a consequence of the mutation is to alter phosphorylation of proteins involved in signal transduction leading to breakdown in cellular signaling pathways mediating phosphatidylserine exposure. An immunoprecipitation method combined with 2‐DE was applied to search for modifications in the expression of phosphorylated polypeptides related to Scott syndrome phenotype. We report here the construction of a B‐lymphoblast subproteomic map comprising of polypeptides observed after immunoprecipitation using antibodies to phosphotyrosine. The polypeptides were identified either by mass fingerprinting, by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and/or by matching with various lymphoid cell 2‐DE maps included in the Laboratoire de Biochimie des Protéines et Protéomique 2‐DE database. A differential analysis was further performed to explore several hundred proteins in Scott B‐lymphoblasts in comparison with control B‐lymphoblasts. Then, image analysis allowed detection of variations between control and Scott syndrome phenotype lymphoblasts. Five spots were specifically found on 2‐DE from Scott syndrome phenotype lymphoblasts, and four only appeared on 2‐DE from control cells. Protein identification was achieved using a combination of mass fingerprinting and peptide identification using LC‐MS/MS.