Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.P rotein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions, subcellular localization, and ultimately cellular functions (1, 2). Deregulation of the signaling network often leads to disease states such as human malignancies, diabetes, and immune disorders. Although many kinases are excellent therapeutic targets, the precise connection between protein kinases and their direct substrates has not been fully elucidated for a majority of protein kinases. Besides classical genetic and biochemical methods, there have been a number of high throughput approaches for the identification of potential kinase substrates. Common methods include in vitro kinase assays using libraries of synthetic peptides (3), phase expression libraries (4), protein/peptide arrays (5-7), or cell extracts (8, 9), but these methods can often be misleading and provide many false positive results. The discovery of physiological substrates for specific protein kinases has remained challenging, even with recent advances in mass spectrometry.Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,1...

Plants can extensively uptake organic contaminants from soil and subsequently transform them into various products. Those compounds containing hydroxyl may undergo direct conjugation with endogenous biomolecules in plants, and potentially be preserved as conjugates, thus enabling overlooked risk via consumptions of food crops. In this study, we evaluated the uptake and metabolism of 2,4-dibromophenol (DBP) by both carrot cells and whole plant. DBP was completely removed from cell cultures with a half-life of 10.8 h. Four saccharide conjugates, three amino acid conjugates, and one phase I metabolite were identified via ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry analysis. The dibromophenol glucopyranoside (glucose conjugate) was quantitated by synthesized standard and accounted for 9.3% of the initial spiked DBP at the end of incubation. The activity of glycosyltransferase was positively related to the production of 2,4-dibromophenol glucopyranoside ( p = 0.02, R = 0.86), implying the role of enzymatic catalysis involved in phase II metabolism.

Background: The number of red blood cells (RBCs) increases significantly in response to high-altitude hypoxic environments, and the RBC microRNA (miRNA) expression pattern is similar to that in whole blood. Studies have shown that miRNA in plasma can act as a circulating hypoxia-associated marker, but the effect of a high-altitude hypoxic environment on RBC-derived miRNAs has not yet been reported.Methods: Blood samples were collected from 20 Han Chinese individuals residing at 500 m (Sichuan Han), 10 migrant Han Chinese citizens residing at 3,658 m (Tibet Han) and 12 native Tibetans, and RBC indices measurements and miRNA sequencing analyses were performed for the three sample groups. The levels of some markedly altered miRNAs at high altitude were subsequently measured from 5 randomly selected samples of each group by real-time PCR. Bioinformatic analyses was performed to determine the potential target genes of selected hypoxia-associated miRNAs.Results: Marked changes of several RBC indices were observed among the Tibet Han population, the Tibetan population and the Sichuan Han population. A total of 516 miRNAs derived from RBCs were initially identified by miRNA sequencing in the three sample groups. Compared with the Sichuan Han population, 49 miRNAs were differentially expressed in the Tibet Han population (17 upregulated and 32 downregulated). 12 upregulated and 21 downregulated miRNAs were observed in the Tibetan population compared with the Sichuan Han population. A total of 40 RBC miRNAs were differentially expressed in the Tibetan population (15 upregulated and 25 downregulated) compared with the Tibet Han population. Two significantly altered miRNAs with the highest expression levels (miRNA-144-5p and miR-30b-5p) were selected for real-time PCR analysis, and the results were consistent with those of miRNA sequencing. Furthermore, bioinformatic analyses showed that some potential target genes of miR-144-5p and miR-30b-5p are involved in the erythroid- hypoxia-, and nitric oxide (NO)-related signaling pathways in response to hypoxia.Conclusion: Our findings provide clear evidence, for the first time, that a high-altitude hypoxic environment significantly affects human RBC miRNA profiles.

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Stem Leydig cells (SLCs), located in the testicular interstitial compartment in the mammalian testes, are capable of differentiating to testosterone-synthesizing Leydig cells (LCs), thus providing a new strategy for treating testosterone deficiency. However, no previous reports have identified and cultured SLCs derived from the pig. The aim of the current study was to isolate, identify, and culture SLCs from pigs. Haematoxylin and eosin staining and immunochemical analysis showed that SLCs were present and that PDGFRα was mainly expressed in the pig testicular interstitium, indicating that PDGFRα was a marker for SLCs in the neonatal pig. In addition, reverse transcription-PCR results showed that SLC markers were expressed in primary isolated LCs, indicating that they were putative SLCs. The putative SLCs were subsequently cultured with a testicular fluid of piglets (pTF) medium. Clones formed after 7 days and the cells expressed PDGFRα. However, no clones grew in the absence of pTF, but the cells expressed CYP17A1, indicating that pTF could sustain the features of porcine SLCs. To summarize, we isolated porcine SLCs and identified their basic characteristics. Taken together, these results may help lay the foundation for research in the clinical application of porcine SLCs.