pr070068e_si_006.pdf (364.97 kB)

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

Download (364.97 kB)
journal contribution
posted on 03.04.2020, 14:57 by Feng Yang, Navdeep Jaitly, Hemalatha Jayachandran, Quanzhou Luo, Matthew E. Monroe, Xiuxia Du, Marina A. Gritsenko, Rui Zhang, David J. Anderson, Samuel O. Purvine, Joshua N. Adkins, Ronald J. Moore, Heather M. Mottaz, Shi-Jian Ding, Mary S. Lipton, David G. Camp, Harold R. Udseth, Richard D. Smith, Sandra Rossie
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