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RBS-ID: Comprehensive identification of RNA-binding sites at single amino acid-resolution

ABSTRACT: We developed RBS-ID, which greatly simplifies the RNA moiety by chemical cleavage, reducing the complexity of MS/MS search space to accurately identify and localize RBS in peptides. RBS-ID comprehensively and robustly identifies RNA-binding sites at both proteome and single protein level.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

SUBMITTER: Jong Woo Bae

LAB HEAD: Jong-Seo Kim

PROVIDER: PXD016254 | Pride | 2020-06-02

REPOSITORIES: Pride

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Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution.

Bae Jong Woo JW Kwon S Chul SC Na Yongwoo Y Kim V Narry VN Kim Jong-Seo JS

Nature structural & molecular biology 20200608 7

RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein-RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically enhance coverage and to reach single amino acid resolution. Moreover, the simple mono-nucleos ...[more]

PMID: 32514175

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Unbiased characterization and systematic profiling of metabolically labeled RNA-binding sites

Project description:Here we introduce pRBS-ID, an amenable platform that enables robust profiling of PAR-crosslinked RBSs. Using tandem mass spectrometry (MS/MS) approaches, we unbiasedly characterize the PAR adducts on peptides, to develop an optimal MS/MS search pipeline that enables robust identification and quantification. Using pRBS-ID, we systematically profile RBSs crosslinked to U or G in PAR-labeled mRNA, which can serve as a rich resource for studying RNA-protein interactions. Especially, pRBS-ID is widely applicable to studies on time-course RNP remodelling, host-virus interactions, and RNP structure prediction.

2021-11-02 | PXD023401 | Pride

LC-MS/MS analysis of an intact synthetic microRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, oligonucleotides with the sequence of mature Drosophila let-7 microRNA, 21 nt in length, were produced synthetically in unmodified and modified (2’-O-methylated at the 3’ uridine) forms. We characterised a 1:1 mixture of both forms of this RNA.

2019-01-10 | PXD012094 | Pride

A translational regulator MHZ9 modulates ethylene signaling in rice

Project description:In this project, we identified a novel RNA-binding protein, MHZ9. And we analyzed the potential proteins interacted with MHZ9 through immunoprecipitation-mass spectrometry (IP-MS). The N-terminal domain of MHZ9 (MHZ9-N) contains a putative RNA splicing and modification domain PRP4. To identify RNA binding sites in the MHZ9-N. We performed XRNAX-IP-MS assay.

2023-07-20 | PXD041240 | Pride

Pytheas SARS-CoV-2 spike protein mRNA sample analysis

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2010-05-27 | E-GEOD-40 | biostudies-arrayexpress

Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions

Project description:In the present paper, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. To prove this three model systems, a) a small synthetic peptide from smB'/B protein incubated with U1snRNA oligonucleotide, b) in vitro reconstituted 15.5K protein with U4 snRNA oligonucleotide and c) native ribonucleoprotein-complexes (RNPs) from S. cerevisiae were used . All protein-RNA complexes were UV irradiated and heteroconjugates cross-linked enriched prior to LC-MS analysis. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks which is observable as a mass increments of 152 Da upon mass spectrometric analysis.

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Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions

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Quantitative photo-crosslinking mass spectrometry reveals protein structure response to environmental changes

Project description:Conformational changes of protein structures depend on their chemical and physical environment. Studying conformational changes depending on environmental parameters is notoriously difficult as many methods of structural biology are affected by parameters like temperature or pH. To make such conformational changes accessible to quantitative crosslinking mass spectrometry (QCLMS) approaches, crosslinking chemistry must be invariant to different conditions. We propose this can be achieved by photo-inducible crosslinkers, which are not influenced by changes in environmental parameters. Here, we introduce a workflow combining photo-crosslinking using 4,4’-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. In this study, we use this novel photo-DIA-QCLMS approach to quantify pH-dependent conformational changes in human serum albumin and cytochrome C. Both proteins show pH dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins, were visualized using hierarchical clustering. For HSA we distinguished the N-F and the N-B form from the native conformation. Additionally, we observed for cytochrome C acidic and basic conformations. In conclusion, photo-DIA-QCLMS distinguished pH-dependent conformers of both proteins.

2019-07-01 | PXD012939 | Pride

Improvements to the rice genome annotation through large-scale analysis of RNA-Seq and proteomics data sets

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