Design and development of nano-proteomics platforms in high-throughput format for biomarker and drug discovery of chronic B cells and tirosin-kinases ( cKIT, ABL1,…) as models

Objetives: 1.-Design and development of beads suspension array (BBAs) suitable for flow cytometry, to determine differential protein expression patterns in aberrant B cells and controls;including phosphorylation levels, protein complexes, etc… 2.-Identification by high-resolution MS/MS ( nano-LC MS/MS) ( OrbiTrap Q-velos) the specific variable regions of BCR in aberrant B cells and controls. 3.-Functional proteomics studies using nanoapproaches: Design and development of NAPPA array with tirosin-quinases ( cKIT, ABL1,BCR-ABL, AKT1,…) and puntual mutants, delections, insertions, etc…. clinically relevant and its evaluation against inhibitors or possible drugs; and comparison between wild-type and mutants. Methodology: A total of 50 individuals will be included ( healthy, B-CLL, B-CLPD). Initially, all individuals will be provided with heathly questionare and immunophenotypic screening will be performed on their peripheral blood samples ( using monoclonal antibodies in four/six combination staining and analyzed by flow cytometry), to detect the possible presence of aberrant B-cells subsets. If present, an extensive molecular characterization will be performed by Nano-proteomics platform: 1.- Differential protein expression profiles by protein beads suspension arrays ( BBAs), 2.-Identification of aminoacidic sequence by nano-LC MS/MS (OrbiTrap Q-velos), 3.-Functional proteomics approaches by NAPPA arrays determining tirosin-quinase activity and how isoforms affect the drug treatment.