title: Applying a targeted label-free approach using LC-MS AMT tags to evaluate changes in protein phosphorylation following phosphatase inhibition.
abstract: 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.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Navdeep
authors.initials: N
authors.lastname: Jaitly
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Jayachandran
authors.forename: Quanzhou
authors.initials: Q
authors.lastname: Luo
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.forename: Xiuxia
authors.initials: X
authors.lastname: Du
authors.forename: Marina A
authors.initials: MA
authors.lastname: Gritsenko
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: David J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: Samuel O
authors.initials: SO
authors.lastname: Purvine
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: Heather M
authors.initials: HM
authors.lastname: Mottaz
authors.forename: Shi-Jian
authors.initials: SJ
authors.lastname: Ding
authors.forename: Mary S
authors.initials: MS
authors.lastname: Lipton
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Harold R
authors.initials: HR
authors.lastname: Udseth
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Sandra
authors.initials: S
authors.lastname: Rossie
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D008387:Marine Toxins; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D010455:Peptides; D010748:Phosphopeptides; C059041:calyculin A