Figure 4.

Determining kinase activity using AAP. (A) Biological duplicates of MCF 10A cells treated with either EGF or lapatinib were analyzed with Western blotting (WB), (B) MS-based in vitro kinase activity assay KAYAK, and (C) AAP peptide-level capture to determine whether AAP preferentially reacts with the “active” form of a kinase. Evidence of EGFR/Mapk pathway activation/inhibition is clear from either WB or KAYAK analyses as changes in Rps6ka1, Akt, and Mapk. However, the levels of these proteins were unchanged in the reaction with the ATP probes, suggesting the probes reacted similarly with the active and inactive form of the kinase. The ATP probes did faithfully reflect the direct inhibition of EGFR by lapatinib. (D) The addition of an extra level of phosphopeptide enrichment following the reaction of ATP probes at the protein level allows quantitative information on the kinase activity to be obtained. Cell lines treated with EGF and lapatinib, respectively, were analyzed using the protein-level AAP enrichment workflow and the peptides labeled with TMT. A small portion (10%) was analyzed directly with LC–MS3 to obtain information on unmodified kinase abundance. The main portion (90%) was enriched for phosphopeptides using TiO₂ before mass spectrometry analysis. Phosphorylations of sites in the activation loop of kinases Mapk1, Mapk3, and Stk24 were all observed to increase and decrease with EGF and lapatinib treatment, respectively, while the unmodified kinase abundance remained unchanged. Error bars represent the minimum and maximum of biological duplicate measurements.