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Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS.

ABSTRACT: BACKGROUND:Phosphoproteins play important roles in a vast series of biological processes. Recent proteomic technologies offer the comprehensive analyses of phosphoproteins. Recently, we demonstrated that surface-enhanced laser desorption/ionization time of flight mass (SELDI-TOF MS) would detect phosphoproteins quantitatively, which was a new application of SELDI-TOF MS. RESULTS:We combined immobilized metal affinity chromatography (IMAC) with SELDI-TOF MS. After SELDI-TOF MS analysis of IMAC-enrichment phosphoproteins from A549 cancer cells, a series of protein peaks at 12.9, 12.8, 12.7 and 12.6 kDa was obtained in a mass spectrum. The peak intensities of these proteins decreased after a phosphatase treatment and, interestingly, they also decreased when the cells were pre-treated with a novel phosphatidylinositol 3-kinase (PI3K) inhibitor, ZSTK474, suggesting that these proteins were ZSTK474-sensitive phosphoproteins. Identity of the phosphoproteins, which were predicted as the multi-phosphorylated forms of 4E-binding protein 1 (4E-BP1) with the aid of TagIdent algorithm, was confirmed by immunoprecipitation and subsequent SELDI-TOF MS analysis. 4E-BP1 is a downstream component of the PI3K/Akt/mTOR pathway and it regulates protein synthesis. We also investigated the effect of ZSTK474 on 4E-BP1 phosphorylation using phospho-specific antibodies. ZSTK474, which have little inhibitory activity for mTOR, inhibited phosphorylation of Ser65, Thr70 and Thr37/46 in 4E-BP1. In contrast, rapamycin, an inhibitor of mTOR, blocked phosphorylation only of Ser65 and Thr70. These results suggest that ZSTK474 and rapamycin inhibited the phosphorylation of 4E-BP1 in a different manner. CONCLUSION:We identified a group of ZSTK474-sensitive phosphoproteins as the multi-phosphorylated form of 4E-BP1 by combining IMAC, SELDI-TOF MS and antibodies.

SUBMITTER: Akashi T

PROVIDER: S-EPMC2670283 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS.

Akashi Tetsuyuki T Yamori Takao T

Proteome science 20090331

<h4>Background</h4>Phosphoproteins play important roles in a vast series of biological processes. Recent proteomic technologies offer the comprehensive analyses of phosphoproteins. Recently, we demonstrated that surface-enhanced laser desorption/ionization time of flight mass (SELDI-TOF MS) would detect phosphoproteins quantitatively, which was a new application of SELDI-TOF MS.<h4>Results</h4>We combined immobilized metal affinity chromatography (IMAC) with SELDI-TOF MS. After SELDI-TOF MS anal ...[more]

PMID: 19331694

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Project description:BackgroundSerum protein profiling seems promising for early detection of breast cancer. However, the approach is also criticized, partly because of difficulties in validating discriminatory proteins. This study's aim is to validate three proteins previously reported to be discriminative between breast cancer cases and healthy controls. These proteins had been identified as a fragment of inter-alpha trypsin inhibitor H4 (4.3 kDa), C-terminal-truncated form of C3a des arginine anaphylatoxin (8.1 kDa) and C3a des arginine anaphylatoxin (8.9 kDa).MethodsSerum protein profiles of 48 breast cancer patients and 48 healthy controls were analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Differences in protein intensity between breast cancer cases and controls were measured with the Mann-Whitney U test and adjusted for confounding in a multivariate logistic regression model.ResultsFour peaks, with mass-to-charge ratio (m/z) 4276, 4292, 8129 and 8941, were found that were assumed to represent the previously reported proteins. M/z 4276 and 4292 were statistically significantly decreased in breast cancer cases compared to healthy controls (p < 0.001). M/z 8941 was decreased in breast cancer cases (p < 0.001) and m/z 8129 was not related with breast cancer (p = 0.87). Adjustment for sample preparation day, sample storage duration and age did not substantially alter results.ConclusionM/z 4276 and 4292 both represented the previously reported 4.3 kDa protein and were both decreased in breast cancer patients, which is in accordance with the results of most previous studies. M/z 8129 was in contrast with previous studies not related with breast cancer. Remarkably, m/z 8941 was decreased in breast cancer cases whereas in previous studies it was increased. Differences in patient populations and pre-analytical sample handling could have contributed to discrepancies. Further research is needed before we can conclude on the relevance of these proteins as breast cancer biomarkers.

Dataset Information

Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS.

Publications

Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS.

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