Fig. 1.

In vitro phosphorylation, enrichment and identification of substrate peptides from cell lysate. To identify a larger number of FLT3 kinase substrates than was previously available, we subjected KG-1 cell lysate to trypsin digestion (Step 1). The tryptic digest was then treated with alkaline phosphatase (Step 2A). Subsequently, the sample was split into two equal parts (Step 2B) prior to in vitro recombinant FLT3 treatment (FLT3-WT, FLT3-D835Y and FLT3-ITD). The samples were enriched with PolyMAC magnetic beads (Step 3) and an aliquot was analyzed on an Orbitrap-Fusion mass spectrometer (Step 4). The mass spectrometer files were uploaded to the Galaxy-P proteomics pipeline for file conversion and ProteinPilot database search (Step 5).