Pulsed SILAC quantitative proteomics profiling to understand the protective role of ginsentide TP1 against hypoxia.
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ABSTRACT: This project used a Pulsed SILAC (pSILAC) proteomics approach to pulsed/chase the incorporation of stable isotope-labeled lysine (13C6 and 15N2 L-lysine) and arginine (13C6 L-arginine) into newly synthesized proteins and profiled de novo protein synthesis in endothelial cells (HUVEC-CS) subjected to hypoxia stress with or without ginsentideTP1.
Project description:SILAC was used to monitor proteome changes to Haloferax volcanii after treatment with oxidizing agent NaOCl. SILAC amino acids used were Lysine(+8) and Arginine(+6). For treatment and control groups 4 replicates were used. In replicates 1 and 3, the control group had supplementation of both light lysine and argninie to the medium and the treatment group was supplemented with both heavy lysine(+8) and arginine(+6). In replicates 2 and 4, the labeling was switched, with the control containing heavy lysine(+8) and arginine(+6) and the treatement was supplemented with light lysine and arginine. After treatment, cells were mixed at a 1:1 ratio (control:treatment) and proteins were extracted and digested with trypsin. For the incorporation test, cells were grown in meduim containing heavy lysine(+8) and argnine(+6) and allowed to grow for 24 hours, then subcultured twice, sequentially after 24 h of growth. To test for incorporation of the heavy amino acids, cells were harvested, proteins extracted, and digested with trypsin.
Project description:Major risk factors for necrotizing enterocolitis (NEC) are formula feeding and prematurity, however, their pathogenic mechanisms are unknown. We found that insufficient arginine/nitric oxide synthesis limits blood flow in the intestinal microvasculature, leading to hypoxia, mucosa damage and NEC in the premature intestine after formula feeding. Formula feeding led to increased intestinal hypoxia in pups at postnatal day 1(P1) and P5, but not in more mature pups at P9. Accordingly, blood flow in the intestinal microvasculature increased after formula feeding only in P9 pups. mRNA profiling revealed that regulators of arginine/nitric oxide synthesis are at higher levels in endothelial cells of the intestine of P9 than P1 pups. Importantly, arginine supplementation increased intestinal microvasculature blood flow, and prevented NEC, whereas an arginine antagonist exacerbated NEC. Our results suggest that balancing intestinal oxygen demand and supply in the premature intestine by modulating arginine/nitric oxide could be used to prevent NEC.
Project description:The purpose of the study is to evaluate the effect of arginine/lysine solution administration on serum potassium levels. A systematic assessment of serum potassium levels will be performed during infusion and up to 24 hours post start of infusion compared to baseline.
Project description:Pseudokinases can modulate activity of protein kinases, among other modes of influencing biological pathways. We tested a hypothesis that knockdown of Tb427tmp.160.4770 (a Pseudokinase) perturbs the Phospho-proteome of T. brucei. To discover changes in the trypanosome phospho-proteome, stable isotope labelling of amino acids in cell culture (SILAC) was performed, followed by enrichment of phosphopeptides with immobilised metal affinity chromatography (IMAC), and phospho-peptide detection/quantitation using mass spectroscopy. Trypanosomes (p2T7-Tb4770) were cultured in heavy (13C6-L-Arginine, 2H4-L-Lysine) or light isotopes (L-Arginine, L-Lysine) for SILAC. Trypanosomes grown in heavy medium were induced with tetracycline for knockdown of Tb427tmp.160.4770. Induced (Tet+, H) and uninduced (Tet-, L) samples were combined and processed together for IMAC and mass spectroscopy
Project description:A long-standing question in developmental and reproductive biology is when the mammalian embryo becomes sufficiently distinct from its oocyte precursor. Myriads of studies examined the messenger RNAs that change during the oocyte-to-embryo transition, whereas proteins have been much less studied, in spite of their greater vicinity to phenotype. In the present study we modified the widely used embryo culture medium KSOM (PMID 12470333, PMID 10859270) to make it apt for our application. We replaced the serum albumin with polyvinylpyrrolidone and also replaced the natural Arginine and Lysine with their “heavy” isotopic variants Arginine 13C 15N and Lysine 13C 15N. Fertilized oocytes were retrieved from oviducts of gonadotropin-primed B6C3F1 females mated to CD1 males, and cultured at 37 degrees Celsius under 5% CO2 in KSOM containing 0.3 mM Arginine 13C 15N and 0.2 mM Lysine 13C 15N, which are the regular concentrations of these two amino acids in KSOM medium (PMID 12470333; PMID 10859270). After 4 days of culture, the embryos of the isotopic group had undergone blastocyst formation just like the control embryos cultured in normal medium. Samples of approx. 500 “heavy”-labeled blastocysts were collected zona-free and subjected to mass spectrometric analysis. The median labeling rate was 83%, ranging from 0% in proteins that did not incorporate any Arginine 13C 15N and Lysine 13C 15N, to 100% in proteins that were completely labeled. Our study demonstrates that a commonly used, chemically defined medium can be adapted for Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and combined with high-resolution mass spectrometry, in a preimplantation embryo setting. This allows to tackle long-standing questions in developmental and reproductive biology, such as the identification of putative maternal (0% labeled), putative embryonic (100% labeled) or shared proteins in live mammalian embryos.
Project description:Whole cell lysate of yeast. Two cultures were grown, one with standard feed and one with heavy SILAC labeled lysine and arginine. Extracts were mixed together in a one to one mixture and run in LC-MS/MS.
Project description:We report the high-throughput profilings of HIF1 and histone modifications in human umbilical vein endothelial cells (HUVEC). By obtaining over two billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of HUVEC under normoxia and hypoxia. We find that HIF1binds to not only to transcriptional starting sites but also enhancer regions and that HIF1 binding sites were overlapped with lysine 4 trimethylatio, monomethylation and lysine 27 acetylation . Finally, we show that chromatin state can change under hypoxia by using chromatin conformational capture assay. This study provides novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1. Examination of HIF1 and 3 different histone modifications in HUVEC under 2 conditions. Related gene expression data is provided in GSE35932.
Project description:Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with mass spectrometry analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and post-translational modifications between cell populations. We have developed and utilised SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans. Arginine metabolism provides important cues for escaping host defences during pathogenesis, which limits the use of auxotrophs in Candida research. Our strategy eliminates the need for engineering arginine auxotrophs for SILAC experiments and allows the use of ARG4 as selectable marker during strain construction. Cells that were auxotrophic for lysine were successfully labelled with both lysine and arginine stable isotopes. We found that prototrophic C. albicans preferentially uses exogenous arginine and downregulates internal production, which allowed it to achieve high incorporation rates. However, similar to other yeast, C. albicans was able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method was developed to correct for the incorporation of heavy proline. In addition, we utilised the developed SILAC labelling in Candida albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14PD.