Project description:Identification of RNA–protein interactions currently requires a quantity and quality of sample that precludes their widespread application, especially for dynamic biological systems or precious samples. Here, we present Orthogonal Organic Phase Separation (OOPS), a new approach to enrich RNA Binding Proteins (RBPs) which is compatible with downstream proteomics and RNA sequencing. The flexibility of OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. We have applied OOPS to both eukaryotic and previously inaccessible prokaryotic models, demonstrating its wide applicability, efficacy and consistency. We observe that the proteins identified include the majority of previously known RBPs, as well as novel groups of RBPs, including membrane proteins. Furthermore, applying OOPS to a cell-cycle arrest model, we can determine dynamic changes in RNA–protein interaction. Taken together, OOPS opens new model-independent, easy-to-implement opportunities to characterize RNA–protein interactions and their dynamic behaviour.
Project description:MS analysis of human cardiac samples from left ventricles of patients undergoing cardiac transplantation due to ischaemic heart failure. Analysis of intracellular protein enriched extracts.
Project description:Resistant starches (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), oppose dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed diets high in fat (19%) and protein (20%) with different forms of digestible starch (low amylose maize starch (LAMS) or low amylose whole wheat (LAW)) or RS (HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference (RNAi)) for 11 wk. A control diet contained 7% fat, 13% protein and LAMS. The aim of this study was to detect changes in the expression of DNA damage and repair genes in response to the above dietary treatments. Distal colon tissues from Sprague Dawley rats fed a variety of diets (see summary) were removed from RNAlater stabilisation reagent (Sigma, Australia) they had been stored in,, placed in 1 ml of TRIzol® Reagent (Invitrogen, Australia) and homogenised usingbeads (mix of 2.5 mm glass and 0.1 - 1.0 mm diameter silicon-zirconian beads) in a MiniBeadbeater-8 (BioSpec Products Inc., USA). Total RNA was extracted (using TRIzol® Reagent manufacturer's instructions) and further purified using RNAeasy mini spin columns (QIAGEN, Australia) with a DNase on-column digestion as per manufacturer's instructions.RNA integrity was checked using a Bioanalyzer 2100 (Agilent Technologies, USA) andquantified using a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA). RNA samples with insufficinet quality and quantity were not assayed. The numbers of rats arrayed from each diet were as follows: CON, 5;LAMS, 7; HAMS, 8; HAMSB, 9; LAW, 6; HAW , 8. This gives a total of 43 arrays.
Project description:Here we identify that drug-resistant BTK mutations occur in distinct enzymatic classes, some of which render BTK enzymatically impaired while conferring novel protein-protein interactions to sustain B-cell receptor (BCR) signaling.
Project description:Tandem mass tags (TMT) enable simple and accurate quantitative proteomics for multiplexed samples by relative quantification of tag reporter ions. Orbitrap™ quantification of reporter ions has been associated with a characteristic notch region in intensity distribution, within which few reporter intensities are recorded. This has been resolved with updated instrument acquisition software, however, 53 % of Orbitrap submissions to PRIDE were generated using the prior software versions. To quantify the impact of the notch on existing quantitative proteomics data, we generate a mixed species benchmark and acquired quantitative data using the outdated software version. Sub-notch intensities are systemically underestimated, leading to over-estimation of the true differences in intensities between samples. However, when summarising reporter ion intensities to higher level features, such as peptides and proteins, few features are significantly affected. The analysis of benchmark dataset indicates that the targeted removal of spectra with reporter ion intensities below the notch is not beneficial for differential peptide or protein testing. Overall, we find the systematic quantification bias associated with the notch is not detrimental for typical proteomics experiments
Project description:To study the meiotic dynamic of the methylation of H3K4, in relation with the transcriptomic regulation, we determine the meiotic time-course of H3K4me3 by ChIP-chip in a mutant deficient for DSB formation. Keywords: ChIP-chip, meiotic time course, histone modification Strain ORD7341 meiotic time-course from 0 to 6h. Immunoprecipitation with rabbit polyclonal anti-triMe H3K4 (Abcam #Ab8580).
Project description:To study the meiotic dynamic of the methylation of H3K4, in relation with the transcriptomic regulation, we determine the meiotic time-course of H3K4me3 by ChIP-chip. Keywords: ChIP-chip, meiotic time course, histone modification Strain ORD7339 meiotic time-course from 0 to 6h. Immunoprecipitation with rabbit polyclonal anti-triMe H3K4 (Abcam #Ab8580).
Project description:To study the meiotic dynamic of the methylation of H3K4, in relation with the transcriptomic regulation, we determine the meiotic time-course of H3K4me3 by ChIP-chip in a mutant deficient for DSB formation in a mutant clb5Delta-clb6Delta Keywords: ChIP-chip, meiotic time course, histone modification Strain ORD6830 meiotic time-course from 0 to 6h. Immunoprecipitation with rabbit polyclonal anti-triMe H3K4 (Abcam #Ab8580).
Project description:To study the meiotic dynamic of the methylation of H3K4, in relation with the transcriptomic regulation, we determine the meiotic time-course of H3 occupancy by ChIP-chip. Keywords: ChIP-chip, meiotic time course, histone modification Strain ORD7339 meiotic time-course from 0 to 6h. Immunoprecipitation with rabbit polyclonal anti H3Cter (Abcam #1791)
Project description:To study the meiotic dynamic of the methylation of H3K4, in relation with the transcriptomic regulation, we determine the meiotic time-course of H3 occupancy by ChIP-chip in a mutant Spo11F. Keywords: ChIP-chip, meiotic time course, histone modification Strain ORD7341 meiotic time-course from 0 to 6h. Immunoprecipitation with rabbit polyclonal anti H3Cter (Abcam #1791)