Project description:This experiment describes the quantitative proteomic profiling of whole-cell, soma, and neuronal projection fractions from day 35 induced neurons (iN) derived from control and ASAH1 knockout human embryonic stem cells. Soma and projection compartments were physically separated using Transwell culture inserts, and triplicate biological replicates were processed using TMTpro 18-plex labeling and high-resolution Orbitrap mass spectrometry with FAIMS. Data were searched against the human proteome with stringent FDR control and reporter ion quantification, enabling compartment-resolved analysis of proteome alterations associated with ASAH1 deficiency.
Project description:This experiment describes a quantitative proteomic analysis of whole-cell lysates from induced neurons (iN) and induced dopaminergic neurons (iDA) differentiated from human embryonic stem cells of three genotypes: control, ASAH1 knockout, and SMPD1 knockout. Triplicate biological samples were prepared and analyzed using TMTpro 18-plex labeling coupled to high-resolution Orbitrap mass spectrometry with FAIMS. Data were processed with target-decoy database searching, stringent FDR control, and reporter ion–based quantification. The resulting dataset enables comparative profiling of proteomic alterations associated with lysosomal dysfunction and neurodegeneration-relevant pathways in distinct neuronal subtypes.
Project description:Longitudinal samples were collected from neonates in the NICU at the Royal Women’s Hospital in Melbourne, Australia. Blood collection occurred by heel stick and was collected on Whatman paper shortly after birth at 25 weeks gestation, one day post birth, and at the equivalent of 28, 32, 36, and 40 weeks gestation.
Project description:The field of proteomics has evolved hand-in-hand with technological advances in LC-MS/MS systems, now enabling the analysis of very deep proteomes in a reasonable time. However, most applications do not deal with full cell or tissue proteomes, but rather with restricted sub-proteomes relevant for the research context at hand or resulting from extensive fractionation. At the same time, investigation of many conditions or perturbations puts a strain on measurement capacity. Here we develop a high throughput workflow capable of dealing with large numbers of low or medium complexity samples and specifically aim at the analysis of 96-well plates in a single day (15 min per sample). We combine parallel sample processing with a modified liquid chromatography platform driving two analytical columns in tandem, which are coupled to a quadrupole Orbitrap mass spectrometer (Q Exactive HF). The modified LC platform eliminates idle-time between measurements and the very high sequencing speed of the Q Exactive HF dramatically reduces required measurement time. We apply the pipeline to the yeast chromatin remodeling landscape, and demonstrate quantification of 96 pull-downs of chromatin complexes in about one day. This is achieved with only 500 µg input material, enabling yeast cultivation in a 96-well format. Our system retrieved known complex-members and the high throughput allowed probing with many bait proteins. Even alternative complex compositions were detectable in these very short gradients. Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved several-fold compared to established workflows. The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.
Project description:The kidney differentiation protocol takes induced pluripotent stem cells through to kidney organoid via directed differentiation in approximately 25 days. The cells are grown in a monolayer in a dish for seven days and are subjected to growth factors before being pelleted on day seven. The organoids then continue to differentiate as a 3D structure, with at least 8 distinct kidney cell types identifiable around day 18. This data has been generated to investigate the reproducibility of the kidney differentiation protocol using RNA sequencing from day 18 organoids. Organoid differentiations were performed in two separate batches separated in time. The resulting RNA-seq data was analysed to identify genes with highly variable expression between batches.
Project description:Comparison of murine colonic mucosal gene expression between postanatal day 90 (P90) to postnatal day 30 (P30) by whole genomic expression microarray. Gene expression profiling of colonic mucosal DNA between P90 and P30 mice. Agilent Technologies two-color labelling kit and genomic hybridization protocol was utilized.
Project description:Stable-isotope labeling strategies are extensively used for multiplex quantitative proteomics. Hybrid isotope labeling strate-gies that combine the use of isotopic mass difference labeling and isobaric tags can greatly increase sample multiplexity. In this work, we present a novel hybrid isotope labeling approach that we termed NHS-ester tandem labeling in one pot (NETLOP). We first optimized 16-plex isobaric TMTpro labeling of lysine residues followed by 2-plex or 3-plex isotopic mTRAQ labeling of peptide N-termini, both of which with commercially available NHS-ester reactive reagents. We then demonstrated the utility of the NETLOP approach by labeling HeLa cell samples and performing proof-of-principle quanti-tative 32-plex and 48-plex proteomic analyses, each in a single LC-MS/MS experiment. Compared to current hybrid isotope labeling methods, our NETLOP approach requires no sample cleanup between different labeling steps to minimize sample losses, induces no retention time shifts that compromise quantification accuracy, can be adapted to other NHS-ester isotop-ic labeling reagents to further increase multiplexity, and is compatible with samples from any origin in a wide array of bio-logical and clinical proteomics applications.
Project description:We aimed at identifying the regulatory network of Aflibercept in the context of pathological neovascularization by using the oxygen-induced retinopathy mouse model as a surrogate of proliferative diabetic retinopathy Total RNA was collected from entire retinal cups dissected from mice subjected to the oxygen-induced retinopathy protocol and subsequently injected with Aflibercept either on postnatal day 13 (collection on day 14) or on postnatal day 13 and 15 (collection on day 17). Non-injected OIR littermates and age-matched mice housed in normoxic conditions were used as controls.
Project description:Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.
Project description:In total, 240 single blastomeres from nine top-quality day-4 embryos frozen at day 3 of development and four fresh top-quality day-4 embryos that had one-cell biopsy on day 3 for preimplantation genetic diagnosis (PGD) were collected. Blastomeres' DNA was amplified using SurePlex DNA Amplification System (BlueGnome, Cambridge, UK) . Array-CGH was carried out using 24Sure Cytochip microarrays following the standard protocol (BlueGnome, www.cytochip.com).