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.
Laboratoire de Biochimie des Protéines et Protéomique (BPP), Université Paris 13, Bobigny, France.
Naïma
N
Imam-Sghiouar
Isabelle
I
Laude-Lemaire
Valérie
V
Labas
Delphine
D
Pflieger
Jean-Pierre
JP
Le Caër
Michel
M
Caron
Danièle Kerbiriou
DK
Nabias
Raymonde
R
Joubert-Caron
D000906:Antibodies / Q000378:metabolism; D001402:B-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006474:Hemorrhagic Disorders / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010641:Phenotype; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry; D040901:Proteomics; D015398:Signal Transduction / Q000502:physiology; D013577:Syndrome
D000906:Antibodies; D020543:Proteome; D019000:Phosphotyrosine