False discovery rate estimation and heterobifunctional cross-linkers

Fischer, Lutz; Rappsilber, Juri

doi:10.1101/239715

Cited by 4 publications

(4 citation statements)

References 26 publications

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“…To create the final list of unique crosslinked peptide pairs, we first concatenated the search results from all crosslinking and sample prep conditions (250:1, 500:1, 750:1, 1000:1 DSSO:augmin molar ratio, and in-gel vs. in-solution digests). Our MetaMorpheus search settings resulted in 1% FDR for both intersubunit and intrasubunit crosslink pairs when calculated using an established formula for target-decoy searches, FDR = (TD−DD)/TT, where TT is the number of target–target crosslink pairs, TD is the number of target–decoy crosslink pairs, and DD is the number of decoy–decoy crosslink pairs 52 , 53 . We then eliminated redundant entries, keeping the highest scoring incidences, and applied a final cutoff below MetaMorpheus q -value of 0.01 (1% FDR).…”

Section: Methodsmentioning

confidence: 99%

Molecular architecture of the augmin complex

Gabel

DeMarco

et al. 2022

Nat Commun

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Accurate segregation of chromosomes during mitosis depends on the correct assembly of the mitotic spindle, a bipolar structure composed mainly of microtubules. The augmin complex, or homologous to augmin subunits (HAUS) complex, is an eight-subunit protein complex required for building robust mitotic spindles in metazoa. Augmin increases microtubule density within the spindle by recruiting the γ-tubulin ring complex (γ-TuRC) to pre-existing microtubules and nucleating branching microtubules. Here, we elucidate the molecular architecture of augmin by single particle cryo-electron microscopy (cryo-EM), computational methods, and crosslinking mass spectrometry (CLMS). Augmin’s highly flexible structure contains a V-shaped head and a filamentous tail, with the head existing in either extended or contracted conformational states. Our work highlights how cryo-EM, complemented by computational advances and CLMS, can elucidate the structure of a challenging protein complex and provides insights into the function of augmin in mediating microtubule branching nucleation.

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Section: Methodsmentioning

confidence: 99%

Molecular architecture of the augmin complex

Gabel

DeMarco

et al. 2022

Nat Commun

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“…Raw files were preprocessed with MaxQuant (v1.5.4.1), using the partial processing until step 5. Resulting peak files (APL format) were subjected to Xi, 17 The respective search databases consisted of a single entry with the sequence of the corresponding protein, that was extracted from the crystal structure PDB file (PDB IDs: 1AO6, 2CRK, 2FRJ). FDR was estimated using XiFDR 18 on 5% residue level with enabled boosting and including only unique peptide-spectra matches (PSMs).…”

Section: ■ Methodsmentioning

confidence: 99%

Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer

2017

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We compared the five different ways of fragmentation available on a tribrid mass spectrometer and optimized their collision energies with regard to optimal sequence coverage of cross-linked peptides. We created a library of bis(sulfosuccinimidyl)suberate (BS3/DSS) cross-linked precursors, derived from the tryptic digests of three model proteins (Human Serum Albumin, creatine kinase, and myoglobin). This enabled in-depth targeted analysis of the fragmentation behavior of 1065 cross-linked precursors using the five fragmentation techniques: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), and the combinations ETciD and EThcD. EThcD gave the best sequence coverage for cross-linked m/z species with high charge density, while HCD was optimal for all others. We tested the resulting data-dependent decision tree against collision energy-optimized single methods on two samples of differing complexity (a mix of eight proteins and a highly complex ribosomal cellular fraction). For the high complexity sample the decision tree gave the highest number of identified cross-linked peptide pairs passing a 5% false discovery rate (on average ∼21% more than the second best, HCD). For the medium complexity sample, the higher speed of HCD proved decisive. Currently, acquisition speed plays an important role in allowing the detection of cross-linked peptides against the background of linear peptides. Enrichment of cross-linked peptides will reduce this role and favor methods that provide spectra of higher quality. Data are available via ProteomeXchange with identifier PXD006131.

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“…To normalize the expression levels of different genes and different samples, the reads were converted to fragments per kilobase of transcripts per million fragments mapped (FPKM) value. The false discovery rate (FDR) is the adjusted P value obtained by multiple hypothesis test correction on the original P value (16). The DEGs in the libraries were defined as having a FPKM value ≥1.5 and an FDR <0.05.…”

Section: Screening Of Degsmentioning

confidence: 99%

Transcriptome sequencing analysis revealed the molecular mechanism of podoplanin neutralization inhibiting ischemia/reperfusion-induced microglial activation

Qian¹,

Qian

Yang³

et al. 2022

Ann Transl Med

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Background: Anti-podoplanin antibody (α-PDPN, clone 8.1.1) reduces microglia-mediated inflammation and decreases cerebral infarct volume in mice with stroke. However, the molecular mechanism by which this occurs is unknown. This study sought to systematically analyze the molecular mechanism of α-PDPN treatment on ischemia/reperfusion (I/R)-injured microglia.Methods: Microglia BV2 cells were pre-cultured with α-PDPN and then exposed to oxygen-glucose deprivation and reoxygenation (OGD-R) insult. The differentially expressed genes (DEGs) underwent a transcriptome sequencing technology analysis, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Quantitative real-time polymerase chain reaction (PCR) was performed to confirm the transcriptional expression of some DEGs. Results:The results showed that α-PDPN downregulated 338 genes and upregulated 340 genes in the BV2 cells. The GO items of the downregulated DEGs mainly involved biological processes, such as the response to the interferon (IFN), lipopolysaccharide-mediated signaling pathway, and the regulation of cell chemotaxis and migration. The upregulated molecular function mainly involved glucocorticoid-receptor binding. Further, the KEGG pathway analysis indicated that the enriched categories for the upregulated DEGs mainly involved the adenosine triphosphate (ATP) binding cassette transporters. However, the interleukin-17 signaling pathway, IFN signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor beta (TGF-ꞵ) signaling pathway, nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathway, cytokine-cytokine receptor interaction, and chemokine signaling pathway were downregulated by the α-PDPN treatment.Conclusions: Numerous inflammation-related signaling pathways were regulated by the α-PDPN treatment in the OGD-R injured BV2 cells. This study provided further insights into the protective mechanism of α-PDPN treatment in ischemic stroke.

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False discovery rate estimation and heterobifunctional cross-linkers

Cited by 4 publications

References 26 publications

Molecular architecture of the augmin complex

Molecular architecture of the augmin complex

Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer

Transcriptome sequencing analysis revealed the molecular mechanism of podoplanin neutralization inhibiting ischemia/reperfusion-induced microglial activation

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