Correction to Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides

Giese, Sven H.; Belsom, Adam; Rappsilber, Juri

doi:10.1021/acs.analchem.7b00046

Cited by 5 publications

(4 citation statements)

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“…Each fraction was analyzed in duplicates. The settings of the mass spectrometer were as follows: Data-dependent mode with 2.5s-Top-speed setting; MS1 scan in the Orbitrap at 120,000 resolution over 400 to 1500 m/z with 250% normalized automatic gain control (AGC) target; MS2 scan trigger only on precursors with z = 3–7+, AGC target set to “standard”, maximum injection time set to “dynamic”; fragmentation by HCD employing a decision tree logic with (Giese et al , 2017) optimized collision energies; MS2 scan in the Orbitrap at a resolution of 60,000; dynamic exclusion was enabled upon a single observation for 60 s. Each LC-MS acquisition took 120 min.…”

Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff

Graziadei

Schildhauer

Spahn

et al. 2022

Preprint

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In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. Nevertheless, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains elusive. We set out to characterize the interactome of full-length Nsp1 and its topology by crosslinking mass spectrometry in order to investigate the role of the N-terminal domain and linker regions in host translational shutoff. We find that these regions are in contact with 40S proteins lining the mRNA entry channel and detect a novel interaction with the G subunit of the eIF3 complex. The crosslink-derived distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit, reporting on the dynamic interface between Nsp1, the ribosome and the eIF3 complex. The significance of the Nsp1-eIF3G interaction is supported by further evidence that Nsp1 predominantly binds to 40-43S complexes. Our results point towards a mechanism by which Nsp1 is preferentially recruited to canonical initiation complexes, leading to selective inhibition of host-translating ribosomes and subsequent mRNA degradation.

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Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff

Graziadei

Schildhauer

Spahn

et al. 2022

Preprint

Self Cite

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“…For sulfo-SDA, the cross-linker mass 82.04 Da and the modifications SDA-loop (+82.04 Da) and SDA-hyd (+100.05 Da) were used. 17 False discovery rate (FDR) estimation was done using xiFDR 18 (v. 1.1.26.58), using either 5% link FDR (without boosting) or a 5% peptide spectrum match (PSM) FDR. The Euclidean cross-link distances within HSA were estimated from mapping the peptide sequences to the three-dimensional structure when possible (PDB: 1AO6(19)).…”

Section: Materials and Methodsmentioning

confidence: 99%

“…The database search with Xi used the following parameters: MS tolerance, 6 ppm; MS2 tolerance, 15 ppm; missed cleavages, 3; enzyme, trypsin; fixed modifications, carbamidomethylation (cm, +57.02 Da); variable modifications, oxidation methionine (ox, +15.99 Da). For sulfo-SDA, the cross-linker mass 82.04 Da and the modifications SDA-loop (+82.04 Da) and SDA-hyd (+100.05 Da) were used . False discovery rate (FDR) estimation was done using xiFDR (v. 1.1.26.58), using either 5% link FDR (without boosting) or a 5% peptide spectrum match (PSM) FDR.…”

Section: Materials and Methodsmentioning

confidence: 99%

Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses

et al. 2019

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Cross-linking mass spectrometry draws structural information from covalently linked peptide pairs. When these links do not match to previous structural models, they may indicate changes in protein conformation. Unfortunately, such links can also be the result of experimental error or artifacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing coelution of the corresponding linear peptides in MS1 spectra, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress noncovalent peptide formations, increasingly disruptive ionization settings can be used, such as in-source fragmentation.

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“…The library was imported as an external library. Protein modifications were defined manually in addition to a default list of modifications in Spectronaut: SDA-loop (82.04 Da), SDA-alkene (68.06 Da), SDA-oxid (98.04 Da), SDA-hydro (100.05 Da), and SDA-N 2 (110.05 Da). , MS1 and MS2 filtering was done following the Spectronaut 12 manual with the following deviations: quantitation tab, interference correction unticked; minor (peptide) grouping, by modified sequence; major group top N unticked and minor group top N ticket (maximum, 6; minimum, 1); minor group quantity, mean precursor quantity, decoy method was set to “scrambled”. Normalized data (local normalization option) with a q -value of 0.01 (comparable to 1% FDR) were exported from Spectronaut to integrate feature-level quantitation data into residue-level data using a top 3 approach.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Photo-crosslinking Mass Spectrometry Revealing Protein Structure Response to Environmental Changes

2019

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Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of 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. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins.

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Correction to Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides

Cited by 5 publications

References 0 publications

SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff

SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff

Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses

Quantitative Photo-crosslinking Mass Spectrometry Revealing Protein Structure Response to Environmental Changes

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