QuaNCAT: quantitating proteome dynamics in primary cells

Howden, Andrew J. M.; Geoghegan, Vincent; Katsch, Kristin; Efstathiou, Georgios; Bhushan, Bhaskar; Boutureira, Omar; Thomas, Benjamin; Trudgian, David C.; Kessler, Benedikt M.; Dieterich, Daniela C.; Davis, Benjamin G.; Acuto, Oreste

doi:10.1038/nmeth.2401

Cited by 162 publications

(150 citation statements)

References 24 publications

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“…Our chemical tagging strategy is compatible with routine MS sample preparation methods such as gel electrophoresis liquid chromatography-mass spectrometry (GeLC-MS), filter-aided sample preparation (FASP) (41,42), and multidimensional protein identification technology (MudPIT) (43), and is easily combined with SILAC, isobaric tag for relative and absolute quantification (iTRAQ), and multiple-reaction monitoring (MRM) quantitative MS methods (44,45). This approach can be complemented with candidate protein methods, such as expression of tagged substrates, to verify the secretion and identify the location of newly identified substrates inside host cells.…”

Section: Discussionmentioning

confidence: 99%

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Mahdavi

Szychowski

Ngo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.proteomics | click chemistry | BONCAT | Yop

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Section: Discussionmentioning

confidence: 99%

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Mahdavi

Szychowski

Ngo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Ni-NTA agarose eluates from triplicate experiments for the isolation of the cross-linked complexes to the respective NosR-His 6ϫ , NorC-His 6ϫ , and NorB-His 6ϫ bait proteins were subsequently evaluated by LC-MS/MS as previously described (55). The eluted proteins with their attached copurified interaction partners were identified by quantitative mass spectrometry (AP-QMS) (56). The LC-MS/MS analyses were performed on a Dionex UltiMate 3000 RSLCnano system connected to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, Germany).…”

Section: Methodsmentioning

confidence: 99%

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

et al. 2016

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Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa. During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO 3؊ ¡ NO 2؊ ¡ NO ¡ N 2 O ¡ N 2 . Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment. IMPORTANCEThe processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.

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“…This has led to a wide range of CuAAC reagents now being commercially available for bioconjugation. Indeed, the CuAAC can be performed site-selectively with complete conversion 34 and has been used in many significant applications, such as the generation of PEGylated proteins 87 , the generation of dual PTM glycoprotein mimics due to its orthogonality to existing cysteine chemistry 34,35 , cellular proteomic analysis (BONCAT) 80 , a quantitative method for primary cell proteomics (QuaNCAT) 88 , and the construction of highly-valent protein nanoparticles 89 . Despite this compatibility, the perceived toxicity of copper has led to the exploration of alternative cycloaddition-type reactions.…”

Section: Review Nature Communications | Doi: 101038/ncomms5740mentioning

confidence: 99%