ProteoWizard: open source software for rapid proteomics tools development

Kessner, Darren; Chambers, Matt; Burke, Robert; Agus, David B.; Mallick, Parag

doi:10.1093/bioinformatics/btn323

Cited by 1,731 publications

(1,501 citation statements)

References 5 publications

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“…The total length of the analysis was 100 min to allow the column re‐equilibration. The raw MS data were converted into Mascot generic files using MSconvert (Kessner et al , 2008). Searches were performed using Matrix Science software using the following parameters: The error tolerance was fixed at 20 ppm for precursor ions and at 0.5 Da for fragment ions.…”

Section: Methodsmentioning

confidence: 99%

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

et al. 2016

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Skp1‐Cul1‐F‐box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma‐associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F‐box and leucine‐rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17‐mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17–Sufu axis in the pathogenesis of medulloblastoma.

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Section: Methodsmentioning

confidence: 99%

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

et al. 2016

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“…Data Analysis-Thermo .raw datafiles from shotgun LC-MS/MS runs were converted to mzml format using Proteowizard version 3.0.5211 (42). The mzml files were searched using MyriMatch version 2.1.132 (43) and MS-GFϩ version 9517 (44) against the human Refseq version 54 database (Sep 25, 2012; 34,590 entries).…”

Section: Lc-ms/ms Analyses-lc-ms/msmentioning

confidence: 99%

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Federspiel

Codreanu

Goyal

et al. 2016

Molecular & Cellular Proteomics

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Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the ␤5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction. To address this possibility, we synthesized an alkynyl analog of CFZ and investigated protein adduction by this analog in HepG2 cells. Using click chemistry coupled with streptavidin based IP and shotgun tandem mass spectrometry (MS/MS), we identified two off-target proteins, cytochrome P450 27A1 (CYP27A1) and glutathione S-transferase omega 1 (GSTO1), as targets of the alkynyl CFZ probe. We confirmed the adduction of CYP27A1 and GSTO1 by streptavidin capture and immunoblotting methodology and then site-specifically mapped the adducts with targeted MS/MS methods. Although CFZ adduction of CYP27A1 and GSTO1 in vitro decreased the activities of these enzymes, the small fraction of these proteins modified by CFZ in intact cells should limit the impact of these offtarget modifications. The data support the high selectivity of CFZ for covalent modification of its therapeutic targets, despite the presence of a reactive electrophile. The approach we describe offers a generalizable method to evaluate the safety profile of covalent protein-modifying

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“…The steps for data analysis are based on our previously published work [25]; OpenMS software is used, with OMSSA as search engine. To apply this workflow with subsequent database search on the raw data that is generated by the manufacturer's instrument software, the raw files are converted into .mzML format [48] by using msconvert of ProteoWizard software [49]. The first step in the workflow is a conventional database search to identify residual non-cross-linked peptides and also non-cross-linked oligonucleotides.…”

Section: Resultsmentioning

confidence: 99%

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

Sharma

Hrle

Kramer

et al. 2015

Methods

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a b s t r a c tRibonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different singleand multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 -RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

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ProteoWizard: open source software for rapid proteomics tools development

Cited by 1,731 publications

References 5 publications

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

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