Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O 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 a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 974991
- Report Number(s):
- PNNL-SA-52241; 24698; 400412000
- Journal Information:
- Journal of Proteome Research, 7(10):4215-24, Journal Name: Journal of Proteome Research, 7(10):4215-24 Journal Issue: 10 Vol. 7
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
99 GENERAL AND MISCELLANEOUS
AFFINITY
ALGORITHMS
CDK1: Cell Division Cycle 2 Protein Isoform 2
CHROMATOGRAPHY
EFFICIENCY
ESTERIFICATION
Environmental Molecular Sciences Laboratory
Erk2
mitogen-activated protein kinase 1
IMAC
immobilized metal-ion affinity chromatography
LC-MS: liquid chromatography coupled with mass spectrometry
LPA
lysophosphatidic acid
LTQ
linear ion trap
MASS SPECTROMETERS
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
PTMs
post-translational modifications
SIM
selected ion monitoring
SPECTRA
STIMULATION
pSer
phosphoserine
pThr
phosphothreonine
pTyr
phosphotyrosine