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.
Yang
Feng
F
Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Jaitly
Navdeep
N
Jayachandran
Hemalatha
H
Luo
Quanzhou
Q
Monroe
Matthew E
ME
Du
Xiuxia
X
Gritsenko
Marina A
MA
Zhang
Rui
R
Anderson
David J
DJ
Purvine
Samuel O
SO
Adkins
Joshua N
JN
Moore
Ronald J
RJ
Mottaz
Heather M
HM
Ding
Shi-Jian
SJ
Lipton
Mary S
MS
Camp
David G
DG
Udseth
Harold R
HR
Smith
Richard D
RD
Rossie
Sandra
S
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*
D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D010455:Peptides; D010748:Phosphopeptides; C059041:calyculin A