A cross-platform toolkit for mass spectrometry and proteomics

Chambers, Matthew; MacLean, Brendan; Burke, Robert; Amodei, Dario; Ruderman, Daniel; Neumann, Steffen; Gatto, Laurent; Fischer, Bernd; Pratt, Brian; Egertson, Jarrett D.; Hoff, Katherine; Kessner, Darren; Tasman, Natalie; Shulman, Nicholas; Frewen, Barbara; Baker, Tahmina A; Brusniak, Mi-Youn; Paulse, Christopher; Creasy, David M.; Flashner, Lisa; Kani, Kian; Moulding, Chris; Seymour, Sean L.; Nuwaysir, Lydia M.; Lefebvre, Brent A.; Kuhlmann, Frank E.; Roark, Joe; Paape, Rainer; Suckau, Detlev; Hemenway, Tina A.; Hühmer, Andreas; Langridge, James; Connolly, B.; Chadick, Trey; Holly, Krisztina; Eckels, Josh; Deutsch, Eric W.; Moritz, Robert L.; Katz, Jonathan E.; Agus, David B.; MacCoss, Michael J.; Tabb, David L.; Mallick, Parag

doi:10.1038/nbt.2377

Cited by 3,199 publications

(2,574 citation statements)

References 14 publications

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“…Raw files were converted to the mzXML format using msconvert (Chambers et al , 2012) and then searched using xQuest (Leitner et al , 2014) against a database containing the sequences of VirB1 to VirB11 and VirD4. False discovery rates (FDR) were estimated using xProphet, and the results were filtered using the following parameters: FDR = 0.05, min delta score = 0.95, MS1 tolerance window = ±10 ppm, Id‐score > 25, minimum number of cleavages per peptide = 3.…”

Section: Methodsmentioning

confidence: 99%

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

et al. 2017

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Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

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Section: Methodsmentioning

confidence: 99%

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

et al. 2017

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“…ProteoWizard [18] was used for peak-picking, filtering out peaks with intensity less than 100 and converting the file to mzML format. Protein search and identifications were performed using MS-GF+ [19] search engine on Homo sapiens (Uniprot TaxID = 9606).…”

Section: Methodsmentioning

confidence: 99%

Profiling plasma extracellular vesicle by pluronic block‐copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion

Zhong

Rosenow

Xiao

et al. 2018

J of Extracellular Vesicle

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Extracellular vesicle (EV)-based liquid biopsies have been proposed to be a readily obtainable biological substrate recently for both profiling and diagnostics purposes. Development of a fast and reliable preparation protocol to enrich such small particles could accelerate the discovery of informative, disease-related biomarkers. Though multiple EV enrichment protocols are available, in terms of efficiency, reproducibility and simplicity, precipitation-based methods are most amenable to studies with large numbers of subjects. However, the selectivity of the precipitation becomes critical. Here, we present a simple plasma EV enrichment protocol based on pluronic block copolymer. The enriched plasma EV was able to be verified by multiple platforms. Our results showed that the particles enriched from plasma by the copolymer were EV size vesicles with membrane structure; proteomic profiling showed that EV-related proteins were significantly enriched, while high-abundant plasma proteins were significantly reduced in comparison to other precipitation-based enrichment methods. Next-generation sequencing confirmed the existence of various RNA species that have been observed in EVs from previous studies. Small RNA sequencing showed enriched species compared to the corresponding plasma. Moreover, plasma EVs enriched from 20 advanced breast cancer patients and 20 age-matched non-cancer controls were profiled by semi-quantitative mass spectrometry. Protein features were further screened by EV proteomic profiles generated from four breast cancer cell lines, and then selected in cross-validation models. A total of 60 protein features that highly contributed in model prediction were identified. Interestingly, a large portion of these features were associated with breast cancer aggression, metastasis as well as invasion, consistent with the advanced clinical stage of the patients. In summary, we have developed a plasma EV enrichment method with improved precipitation selectivity and it might be suitable for larger-scale discovery studies.

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“…HPLC-purified peptides were analyzed with nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS), using an Acquity LC instrument (C18 column with a 75 m ϫ 250-mm, 1.7-m particle size; Waters) coupled to a Thermo LTQ Orbitrap Velos mass spectrometer (resolution of 60,000 full-width half maximum at mass/charge 400, Top 20, collision-induced dissociation), using a gradient of 1-40% acetonitrile for 60 minutes at a flow rate of 250 nl/minute, as described previously (18). Peak lists containing MS/MS spectra were generated using MSConvert (19), and these lists were then searched, using Mascot version 2.3.01 (http://www.matrixscience.com), against the Swiss-Prot protein database with the taxonomy restriction "mouse" (16, Peptide synthesis and peptide binding assay in T2.B27 cells. Four identified peptides (LRNQSVFNF, SR-LRNQSVFNF, GRLAAIVAK, and GRLAAIVAK-QV), 1 predicted N-terminally extended 11-mer peptide (LL-GRLAAIVAK), and 2 artificially mutated peptides (SA-LRNQSVFNF and LR-GRLAAIVAK) were synthesized using Fmoc chemistry and then verified with matrix-assisted laser desorption ionization MS.…”

Section: Methodsmentioning

confidence: 99%

Critical Role of Endoplasmic Reticulum Aminopeptidase 1 in Determining the Length and Sequence of Peptides Bound and Presented by HLA–B27

Chen

Fischer

Peng

et al. 2014

Arthritis & Rheumatology

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Objective. HLA-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are the two strongest genetic factors predisposing to ankylosing spondylitis (AS). A key aminopeptidase in class I major histocompatibility complex presentation, ERAP1 potentially contributes to the pathogenesis of AS by altering HLA-B27 peptide presentation. The aim of this study was to analyze the effects of ERAP1 on the HLA-B27 peptide repertoire and peptide presentation to cytotoxic T lymphocytes (CTLs).Methods. ERAP1-silenced and -competent HeLa.B27 and C1R.B27 cells were isotope-labeled, mixed, lysed, and then immunoprecipitated using W6/32 or ME1 antibodies. Peptides bound to HLA-B27 were eluted and analyzed by tandem mass spectrometry. Selected peptides were synthesized and tested for HLA-B27 binding ability. The effect of ERAP1 silencing/ mutation on presentation of an immunodominant viral HLA-B27 epitope, KK10, to CTLs was also studied.Results. In both HeLa.B27 and C1R.B27 cells, the proportion of 9-mer HLA-B27-bound peptides was decreased by ERAP1 silencing, whereas the percentages of longer peptides (11-13 mer) were increased. Surprisingly, following ERAP1 silencing, C-terminally extended peptides were readily identified. These were better able to bind to HLA-B27 than were N-terminally extended peptides lacking an arginine at position 2. In both HeLa.B27 cells and mouse fibroblasts expressing HLA-B27, the absence of ERAP1 reduced peptide recognition by HLA-B27-restricted KK10-specific CTLs following infection with recombinant vaccinia virus or transfection with minigenes expressing KK10 precursors. Presence of an AS-protective variant of ERAP1, K528R, as compared to wild-type ERAP1, reduced the peptide recognition by KK10 CTLs following transfection with extended KK10 minigenes.Conclusion. These results show that ERAP1 directly alters peptide binding and presentation by HLA-B27, thus demonstrating a potential pathogenic mechanism in AS. Inhibition of ERAP1 could potentially be used for treatment of AS and other ERAP1-associated diseases.The human leukocyte antigen HLA-B27 is very strongly associated with development of the common inflammatory rheumatic disease ankylosing spondylitis (AS). However, the pathogenic mechanism remains unclear. Recent genome-wide association studies have identified additional AS-associated genes, of which the strongest association is with endoplasmic reticulum aminopeptidase 1 (ERAP1) (1). The strong genetic association of AS with ERAP1 single-nucleotide polymorphisms has been confirmed in different populations (2-7).

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A cross-platform toolkit for mass spectrometry and proteomics

Cited by 3,199 publications

References 14 publications

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Profiling plasma extracellular vesicle by pluronic block‐copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion

Critical Role of Endoplasmic Reticulum Aminopeptidase 1 in Determining the Length and Sequence of Peptides Bound and Presented by HLA–B27

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