Defining mitogen-activated protein kinase pathways with mass spectrometry-based approaches.

Mitogen-activated protein kinases are a group of ubiquitously expressed kinase pathways that have been conserved from yeast through humans. They control a large number of critical cell functions. Identification of targets of those kinases is necessary to define signal transduction pathways that lead to cell responses. The application of a number of mass spectrometry-based techniques to the identification of phosphoproteins is reviewed. A new proteomic approach is described for the identification of the downstream targets of specific kinases that combines phosphorylation of cell lysates in in vitro kinase reactions by active recombinant kinase with protein separation by two-dimensional (2D) gel electrophoresis or SDS-PAGE and phosphoprotein identification by MALDI-TOF mass spectrometry or by phosphopeptide enrichment and tandem mass spectrometry. The results suggested that a combination of multiple approaches will be required to fully identify phosphoproteomes.