NMR resonance assignments of the FinO-domain of the RNA chaperone RocC

Eidelpes, Reiner; Kim, Hyeong Jin; Glover, J. N. Mark; Tollinger, Martin

doi:10.1007/s12104-020-09983-2

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…The IMER was utilized to obtain sequence confirmation by peptide mapping of three different in-house recombinantly produced proteins. As the first protein studied, 0.46 μg of samples of the RNA chaperone FinO-domain RocC (produced as described by Eidelpes et al 52 ) in 0.8% FA and 1 mM NH 4 HCO 3 was digested online at 10 μL·min –1 , trapped on a 4.0 × 3.0 mm C18 column and desalted (8 min in total). The average pressure during this process was 85 bar.…”

Section: Resultsmentioning

confidence: 99%

3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis

Rainer

Egger²,

Zeindl

et al. 2022

Anal. Chem.

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Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 μm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (μIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the μIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed μIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.

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Section: Resultsmentioning

confidence: 99%

3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis

Rainer

Egger²,

Zeindl

et al. 2022

Anal. Chem.

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“…Their role as new RNA matchmakers, mechanistically distinct from Hfq, is being intensively studied, but the molecular details of the interplay between ProQ and its natural RNA ligands only begin to emerge (Melamed et al, 2020). Structural and functional studies highlighted the importance of the conserved N-terminal FinO-like domain, which harbors most of the RNA-binding activity and places ProQ into a larger family of FinO-like RNA chaperones (Chaulk et al, 2010(Chaulk et al, , 2011Glover et al, 2015;Gonzalez et al, 2017b;Eidelpes et al, 2020;Gerovac et al, 2020Gerovac et al, , 2021Immer et al, 2020;Pandey et al, 2020;Stein et al, 2020). However, the role of the C-terminal "Tudor-like" domain (Figure 6), that contributes to the RNA chaperone activities of ProQ (Chaulk et al, 2011;Gonzalez et al, 2017b), does not cease to intrigue researchers.…”

Section: Class Iib: New Ribosome Assembly Factorsmentioning

confidence: 99%

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

Gerovac

Vogel

Smirnov

2021

Front. Mol. Biosci.

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Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.

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“…All samples were stored in aliquots of 100 μL at −20 °C, and pepsin solutions were prepared the day before use. Inhouse produced and purified recombinant RocC 24−126 protein samples were prepared as described by Eidelpes et al 28 and desalted using PD-10 prepacked desalting columns (GE Healthcare, Chicago, USA) according to the manufacturer's protocol. MS grade 5 mM NH 4 HCO 3 solution was used as the elution buffer, resulting in a 0.26 mg•mL −1 protein solution (RocC1).…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Microdroplet Mass Spectrometry Enables Extremely Accelerated Pepsin Digestion of Proteins

Rainer

Eidelpes

Tollinger

et al. 2021

J. Am. Soc. Mass Spectrom.

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In microdroplets, rates of chemical or biomolecular reactions can exceed those in the bulk phase by more than a million times. As electrospray ionization-based mass spectrometry (MS) involves the formation of charged microdroplets, reaction acceleration and online MS monitoring of reaction products can readily be performed at the same time. We investigated accelerated enzymatic reactions in microdroplets and focused on the proteolytic enzyme pepsin. Electrosonic spray ionization (ESSI) was utilized for developing the ultrarapid pepsin in-spray digestion of two different proteins, cytochrome c and RocC, at low pH values. The optimization of the protein digestion aimed at achieving maximum sequence coverage for the analyzed proteins. Furthermore, carefully designed control experiments allowed us to unambiguously prove that enzymatic protein cleavage almost exclusively occurs within the spray at a millisecond time scale and not prior to microdroplet generation.

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NMR resonance assignments of the FinO-domain of the RNA chaperone RocC

Cited by 3 publications

References 16 publications

3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis

3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

Microdroplet Mass Spectrometry Enables Extremely Accelerated Pepsin Digestion of Proteins

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