Patent EP-2124051-A1 (EP)
Inventor: AEBERSOLD RUEDI
IPC: G01N33/68
Filed: 2008-05-23
Abstract: The invention relates to a new method for the detection and identification of direct and/or indirect phosphorylation/dephosphorylation sites of protein kinases and phosphatases as well as to generate phosphorylation profiles of cells, tissues and body fluids at different physiological states. The such identified phosphorylation/ dephosphorylation sites and phosphorylation profiles and their diagnostic markers can be used to monitor kinase and/or phosphatase activity, to support drug target discovery (e.g. by identifying which kinases/pathways are disregulated in a given diseased state), to classify diseased tissues for optimal treatment and for drug development (e.g. kinase inhibitor screens, off target effect assessment, pharmacokinetics,...). It furthermore comprises the generation of corresponding specific mass spectrometry assays for the detection an quantification of a list or library of identified specific kinase and/or phosphatase targets. Furthermore it pertains to the use of such a selective diagnostic marker and/or of such a mass spectrometry assay, respectively, for the measurement and/or analysis of kinase and/or phosphatase activity, preferentially under differentially influenced conditions by comparison of proteome samples subjected to and not subjected to physiologically/biologically/chemically active compounds. The method in particular includes the following steps: (1a) selective isolation of phosphopeptides from a digest of a reference (sub)proteome to form at least one reference phosphopeptide isolate; (1b) selective isolation of phosphopeptides from a cell state which yields the (sub)proteome of interest (POI). The cell states and therefore the POI can comprise first, a proteome in which a defined kinase or phosphatase gene was knocked-out, knocked down, over expressed or the kinase or phosphatase was inhibited or activated or was temperature sensitive or lacked its activity (kinase dead) or second, a proteome in which a kinase and/or phosphatase or signalling pathway was disregulated (e.g. cancer cells/tissue) or third, a proteome from diseased tissue in which one or several kinases or phosphatases or signalling pathways were disregulated to form at least one phosphopeptide isolate of interest; (2) generation of individual LC-MS(/MS) maps (phosphoproteome patterns) for each phosphopeptide isolate using quantitative (label-free) mass spectrometry; (3) correlation and comparison of the at least one phosphoproteome pattern of the reference phosphopeptide isolate with the at least one phosphoproteome pattern of the POI phosphopeptide isolate and determination of phosphopeptide features showing differential behaviour between the wild-type and the POI phosphopeptide isolate; (4) collecting data and determination of correspondingly regulated phosphopeptide features according to steps (1b)-(3) for at least one further or several or all possible POI(s) (as defined under 1b) (5) the determination of at least one or a list of regulated phosphopeptide feature(s) uniquely regulated by a specific kinase or phosphatase, between cell states (POIs) yielding its selective diagnostic marker(s).