Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.

BackgroundAs osteoarthritis (OA) is a highly heterogeneous disease in terms of progression, establishment of prognostic biomarkers would be highly beneficial for treatment. The present study was performed to identify novel biomarkers capable of predicting the progression of knee OA.MethodsA total of 69 plasma samples (OA patients undergoing radiographic progression, n = 25; nonprogression, n = 33; healthy donors, n = 11) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and ion peaks of interest were identified by liquid chromatography and matrix-assisted laser desorption/ionization (MALDI)-TOF MS. The identities of these proteins were further validated by immunoprecipitation combined with SELDI-TOF MS analysis.ResultsSELDI-TOF MS analysis indicated that the intensities of 3 ion peaks differed significantly between progressors and nonprogressors. Subsequent analyses indicated that these peaks corresponded to apolipoprotein C-I, C-III, and an N-terminal truncated form of transthyretin, respectively. The identities of these proteins were confirmed by the loss of ion peaks in SELDI-TOF MS spectra by immunoprecipitation using specific antibodies for the respective proteins.ConclusionsThree potential biomarkers were identified whose serum levels differed significantly between OA progressors and nonprogressors. These biomarkers are expected to be prognostic biomarkers for knee OA and to facilitate the development of novel disease-modifying treatments for OA.

Increased release of thromboxane A 2 (TXA 2 ) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA 2 analogue (STA 2 ) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA 2 stimulated Cl − secretion in a concentration-dependent manner with an EC 50 of 0.06 µM. The STA 2 -induced Cl − secretion was significantly inhibited by ONO-3708 (10 µM), a specific TXA 2 receptor antagonist. The effect of STA 2 (0.3 µM) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA 2 -induced Cl − secretion in the human colonic mucosa (IC 50 value 1.18 µM). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA 2 -induced Cl − secretion was significantly inhibited by 8-bromo-2 -monobutyryladenosine-3 ,5 -cyclic monophosphorothioate (50 µM), a membrane-permeant cAMP antagonist. STA 2 (0.3 µM) significantly increased the intracellular cAMP levels and the short-circuit current via TXA 2 receptor in a human colonic cell line. These results suggest that the TXA 2 -induced Cl − secretion in the colon is mediated via the cAMP pathway in addition to the Ca 2+ -calmodulin pathway which was previously reported.

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