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Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

ABSTRACT: Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

PROVIDER: PRJNA875378 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR21375816.fastq.gz	Fastqsanger.gz
	SRR21375817.fastq.gz	Fastqsanger.gz
	SRR21375818.fastq.gz	Fastqsanger.gz
	SRR21375819.fastq.gz	Fastqsanger.gz
	SRR21375820.fastq.gz	Fastqsanger.gz

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Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

Project description:New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to ~500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

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Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

Project description:21 total proteome samples from E. coli MG1655 wild type fractionated by size exclusion chromatography resulting in 20 fractions. A lysate sample is given as input control.

2022-11-15 | PXD036475 | Pride

Cyanobacterial identification by native mass spectrometry

Project description:Cyanobacterial identification by native mass spectrometry

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Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq).

| S-EPMC9808575 | biostudies-literature

System-wide profiling of protein-complexes via size exclusion chromatography– mass spectrometry (SEC-MS)

Project description:In living cells most proteins are organized in stable or transient functional assemblies known as protein complexes, which control a multitude of vital cellular processes such as cell cycle progression, metabolism, and signal transduction. System-wide workflows for analysis of protein complexes using mass spectrometry based proteomics consists of a biochemical fractionation methods which take advantage of biophysical features such as charge or mass of protein complexes. In this project, we apply size exclusion chromatography to investigate protein assemblies from a Jurkat whole cell lysate. We introduced a fast and robust in-plate sample preparation workflow which allows for replicates to be performed with fewer technical hurdles and with a high degree of reproducibility) We acquired the data in SWATH-mode in combination with a high-throughput LC-system (EVOSEP one). The bioinformatics pipeline builds on the OpenSWATH workflow combined with CCprofiler, a tool for statistical scoring of protein co-elution profiles. This method enabled us to identify more than 100 protein complexes from mammalian cells within one day of measurement.

2022-05-06 | PXD018033 | Pride

MMP qualitative and quantitative mass spectrometry

Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2. Keywords: isotope labeling mass spectrometry, quantitative proteomics

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SEC-SWATH-MS HEK293 - Complex-centric proteome profiling by SEC-SWATH-MS

Project description:Increasingly, biochemical co-fractionation-based approaches are used to study interactomes and protein complexes at high throughput. The devised methods facilitate the qualitative assignment and prediction of hundreds of putative cellular assemblies in one experiment and without dependency on genetic engineering to introduce affinity tags. The present dataset consists of a native proteome extracted by mild lysis from the HEK293 cell line and fractionated into 80 fractions along high resolution size exclusion chromatography, and each fraction analyzed via bottom-up SWATH mass spectrometry. In multi-tiered targeted analysis, from this fragment ion level chromatographic data, quantitative complex assembly information of the proteome is reconstructed in three steps, (i) peptide-centric detection of peptide analytes within each SEC fraction based on fragment ion co-elution groups; (ii) protein-centric detection of protein elution in SEC based on fragment peptide co-elution groups in SEC; (iii) complex-centric detection and quantification of protein complexes and -variants based on component subunit protein co-elution groups in SEC. The data delineate a global picture of quantitative complex formation within a human proteome, including deconvolution of novel subversions and assembly intermediates of critical cellular complexes with essential functions.

2019-01-11 | PXD007038 | Pride

Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging

Project description:Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

2024-10-09 | GSE279139 | GEO

Self-packed size-exclusion columns enable versatile high-throughput native, top-down, and ion-mobility-mass spectrometry studies on proteins and complexes.

Project description:Native MS (nMS) is a key structural biology technique that makes it possible to study intact proteins and their interactions. Unfortunately, non-volatile salts are incompatiblewith nMS, which demands a laborious desalting procedure. Non-denaturing size exclusion chromatography (SEC) allows both rapid desalting and separation and has previously been explored for nMS automation. However, SEC at conventional scale requires rather large sample amounts as well as harsh ESI conditions, which can cause protein unfolding. Capillary LC allows softer conditions; however, the few commercially available SEC columns appropriate for this flow rate are prohibitively expensive for many laboratories. Existing protocols for packing buffer exchange columns rely on specialized equipment and/or result in columns that have limited capacity for size-based protein separation. Here, we present self-packed miniaturized SEC columns with different stationary phases and customizable dimensions. The columns, produced via slurry packing with an ordinary LC pump were used across a range of samples in several applications including nMS, top-down MS (TDMS), ligand screening, and ion mobility (IM)-MS. Native separation allowed acquisition of data from samples containing more than one protein. We acquired native TDMS data of 3 proteins in 12 minutes, with up to 47% sequence coverage. IM-MS of alpha-synuclein at different charge states was measured in ca. 60 minutes (including calibrants), with results that match the literature. Finally, we used SEC-nMS to rapidly screen proteolysis-targeting chimera candidates and performed collision-induced unfolding (CIU) of a PROTAC-induced ternary complex. Through this work, we highlight the potential of SEC to support developments in structural MS.

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