Project description:Here, we introduce a simple method, termed CHOPS, for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well-suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. This work delineates a practical technology for identifying protease substrates, which we anticipate will complement available “N-terminomic” approaches.
Project description:Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5,500 cysteine sites across ~3,000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.
Project description:Identification of cell types in the interphase between muscle and tendon by single-nuclei RNA-seq of three human semitendinous muscle-tendon biopsies. With special focus on the myotendinous junction specific myonuclei, transcripts were identified and confirmed to myotendinous junction with immunofluorescence.
Project description:Mass spectrometry-based proteomics studies have enabled identification and quantification of thousands of proteins from biological samples. High molecular weight abundant proteins are readily sampled in global proteomics studies compared to low molecular weight less abundant proteins. Although extensive fractionation can facilitate identification of low molecular weight less abundant proteins, it requires additional infrastructure, mass spectrometry time and labour. There is a need for a simple method that can deplete high molecular weight proteins and enrich for low molecular weight proteins to improve their coverage in proteomics studies. We developed a simple method that depletes high molecular weight proteins from various biological samples including cell lines, tissues, and serum. Using this strategy, we demonstrate identification of proteins that are often underrepresented in proteomics datasets. This approach also enabled identification of low abundant serum proteins that are often not detected using immunodepletion strategy. We also identified several novel proteins encoded by annotated non-coding regions in the human genome including lncRNAs and UTRs. Our approach can complement existing proteomics workflows to increase detection and coverage of low molecular weight less abundant proteins.
Project description:Peptides from low abundance non-canonical proteins encoded by the human genome are presented by the major histocompatibility complex and serve as potential neoantigens or therapeutic targets. However, their prevalence in the genome is unclear. We identified several non-canonical proteins produced by breast cancer cell lines using proteogenomics approach. Although these proteins were detectable, the transcripts and corresponding proteins showed low abundance and inconsistent expression pattern. Targeting the nonsense-mediated decay pathway by UPF1-knockdown increased the levels of both non-coding transcripts and non-canonical proteins, suggesting they are subjected to degradation by conserved quality control mechanisms in cells. We also observed increased expression of unannotated transcripts and human leukocyte antigen transcripts associated with antigen presentation. These observations suggest that UPF1 has a role in regulating or suppressing transcriptional noise and that modulating the expression level of UPF1 could expand the reservoir of neoantigens and increase neoantigen presentation, potentially augmenting immunotherapeutic responses in cancers.
Project description:We investigate if the differences in phenotype and transcriptome over age might be explained by an underlying change on the epigenetic level. We performed single-cell ATAC sequencing using the 10x Chromium platform. We profiled 2259 nuclei prepared from 3 young liver tissues and 2490 nuclei from 3 old liver tissues.
Project description:We investigate if the differences in phenotype and transcriptome over age might be explained by an underlying change on the epigenetic level. We performed single-cell ATAC sequencing using the 10x Chromium platform. We profiled 4838 nuclei prepared from 3 young liver tissues and 3361 nuclei from 3 old liver tissues.
Project description:The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. We developed a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. To generate the HyPro-seq dataset reported here, fixed and permeabilized human cells were hybridized with digoxigenin-labeled oligonucleotide probes against noncoding RNAs 45S, NEAT1 or PNCTR and transcripts co-localizing with these RNAs were biotinylated in situ using a custom-engineered HyPro enzyme containing a digoxigenin-binding domain. Biotinylated RNAs were then purified and sequenced using the NextSeq 500 Illumina platform.
Project description:HBEC-5i (human brain endothelial cells) grown to confluence in 6-well tissue culture plates. Cocultures were either left unstimulated or incubated overnight with recombinant TNF?. HBEC were then washed in PBS and incubated with PLT (platelets), pRBC (P.falciparum-parasited red blood cells) or NRBC (normal red blood cells) according to the experimental conditions. Then HBEC were washed three times with PBS, and were harvested at 0 and 5h coculture. Each experimental condition was realized in triplicate.