Project description:Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesion in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2¢-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol and its subsequent deamination also yields Tg. However, its distribution in the human genome remains unknown. Here, we presented a DNA-protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach is capitalized on the specificity of a DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in HEK293T cells and the isogenic NTHL1 and NEIL1 double knock out cell lines. We found that Tg is depleted in genomic regions associated with active transcription, but enriched at the nucleosome-binding sites, especially at heterochromatin sites. Collectively, our approach allows for comprehensive analysis of Tg in the human genome and provides a robust tool for future functional studies of Tg in DNA. It can be envisaged that the method can be adapted for mapping other modified nucleosides in genomic DNA in the future.
Project description:Transcriptome-wide profiling of RNA-binding by IGF2BP1 and IGF2BP3 in the K562 cell line. Binding sites are characterized by T-to-C cross-linking mutations, depth and location on transcript
Project description:Transcriptome-wide profiling of RNA-binding by four RBPs in v-Abl transformed 220-8 pro-B mouse cell line. Binding sites are characterized by T-to-C cross-linking mutations, depth, location on transcript, and overlap/exclusivity across different RBPs.
Project description:Mass spectrometry analysis in combination with the site-specific chemical cross-linking has emerged as a powerful method in study of three-dimensional structure of protein complex and in mapping of protein-protein interactions (PPIs). Even though in vitro cross-linking experiments have been widely applied to investigate the specific interactions of a bait protein and its targets, the measurement of in vivo protein tertiary structure and PPIs has been problematic and strenuous due to the dynamic nature of the biological systems and a lower number of cross-linked peptides that can be isolated via MudPIT (Multidimensional Protein Identification Technology) for mass spectrometry analysis. Using Arabidopsis thaliana as a model multicellular eukaryotic organism, we have attempted to develop an improved in vivo chemical cross-linking and mass spectrometry (or IPXL-MS) workflow, which aims at optimizing the in vivo cross-linking conditions, establishing of a MudPIT procedure for enrichment of cross-linked peptides, and developing an integrated software program to identify the in planta chemical cross-linked peptides, by which three pairs of in vivo cross-linked peptides of high-confidence has been identified twice from two independent biological replicates. This work has demarked a beginning of alternative proteomic approach in study of in vivo protein tertiary structure and PPIs in higher plants. This in vivo cross-linking approach may be applied into other model multicellular organisms, such as mouse, for molecular systems biological research.
Project description:In this study, we used bottom-up proteomics and cross-linking MS (XL-MS) to study the composition and structure of soluble membrane attack complex (sMAC). For bottom-up proteomics sMAC was digested using trypsin and analyzed on Orbitrap Fusion Lumos. For the cross-linking analysis sMAC was cross-linked using DMTMM or DSS. The cross-linked proteins were digested using trypsin and the data was acquired using an Ultimate 3000 system coupled on-line to an Orbitrap Fusion. Proteomics raw data was searched using MaxQuant and cross-linking raw data was searched using pLink.
Project description:Cross-linking mass spectrometry data of cross-linked antibody to human leukocyte antigen (HLA-A*11:01). For antibodies 2E3, 1E3 and 3H10.
Project description:Pull-down of poly(A)-mRNA cross linked proteins using two cross-linking methods (conventional cross-linking and PAR-cross-linking) to identify all mRNA-binding proteins (GO:0003729). The provided data is quantitative proteomic data for comparison of cross-linking and control samples.
Project description:In vivo cross-linking and ribonucleoprotein-immunopurification experiments followed by microarray analysis of bound RNAs (X-RIP-chip). Cells expressing recombinant tandem-affinity purification (TAP)-tagged Trf4 protein were cross-linked with formaldehyde, and Trf4-containing ribonucleoprotein complexes were recovered by affinity selection on IgG-coupled beads (see linked protocol). As a control for non-specifically enriched RNAs, the same experiment was done with untagged WT cells and with cells expressing Fpr1-TAP, a peptidyl-prolyl-cis-trans-isomerase not expected to bind RNA. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Antigenic peptide used in IP: Protein A derivative Computed
Project description:In vivo cross-linking and ribonucleoprotein-immunopurification experiments followed by microarray analysis of bound RNAs (X-RIP-chip). Cells expressing recombinant tandem-affinity purification (TAP)-tagged Trf4 protein were cross-linked with formaldehyde, and Trf4-containing ribonucleoprotein complexes were recovered by affinity selection on IgG-coupled beads (see linked protocol). As a control for non-specifically enriched RNAs, the same experiment was done with untagged WT cells and with cells expressing Fpr1-TAP, a peptidyl-prolyl-cis-trans-isomerase not expected to bind RNA. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Antigenic peptide used in IP: Protein A derivative