Applying a Targeted Label-Free Approach Using LC−MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

To identify phosphoproteins regulated by the
phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of
changes in protein phosphorylation on extracts from HeLa
cells treated with or without calyculin A, a potent PPP
enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity
chromatography and targeted tandem mass spectrometry
was employed to discover and identify signatures based
upon distinctive changes in abundance. Overall, 232
proteins were identified as either direct or indirect targets
for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of
both phosphorylation site and protein. These include
phosphorylation sites within signaling proteins such as
p120 Catenin, A Kinase Anchoring Protein 8, JunB, and
Type II Phosphatidyl Inositol 4 Kinase. These data can be
used to define underlying signaling pathways and events
regulated by the PPP family of S/T phosphatases.
Keywords: label-free quantitation • targeted MS/MS • AMT tag
pipeline • comparative phosphoproteomics • immobilized metal
ion affinity chromatography (IMAC) • mass spectrometry • 20 μm
ID monolithic column • phosphoprotein phosphatase (PPP) family
• Ser/Thr protein phosphatase • calyculin A