Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography.
Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.
Ding
Shi-Jian
SJ
Department of Pathology/Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Wang
Yingchun
Y
Jacobs
Jon M
JM
Qian
Wei-Jun
WJ
Yang
Feng
F
Tolmachev
Aleksey V
AV
Du
Xiuxia
X
Wang
Wei
W
Moore
Ronald J
RJ
Monroe
Matthew E
ME
Purvine
Samuel O
SO
Waters
Katrina
K
Heibeck
Tyler H
TH
Adkins
Joshua N
JN
Camp
David G
DG
Klemke
Richard L
RL
Smith
Richard D
RD
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects*; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology*; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism
D008246:Lysophospholipids; D008670:Metals; D010103:Oxygen Isotopes; D010748:Phosphopeptides; C032881:lysophosphatidic acid
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