Universal sample preparation method for proteome analysis

Wiśniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna; Mann, Matthias

doi:10.1038/nmeth.1322

Cited by 7,010 publications

(5,854 citation statements)

References 20 publications

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“…ACN was then evaporated under vacuum and the remaining solution was centrifuged at 200 g for 5 min. The supernatant was collected and processed for mass spectrum analysis using a previously established FASP (Filter assisted sample preparation) protocol 33 , a process that includes reduction, alkylation, and digested with trypsin. The peptides were then extracted, lyophilized, and resuspended in 2% acetonitrile/98% (0.1% formic acid).…”

Section: Methodsmentioning

confidence: 99%

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

et al. 2017

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Immunotherapy holds tremendous promise for improving cancer treatment1. Administering radiotherapy with immunotherapy has been shown to improve immune responses and can elicit an “abscopal effect”2. Unfortunately, response rates for this strategy remain low3. Herein, we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NPs formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent upon NP surface properties. We showed that AC-NPs deliver tumor specific proteins to antigen-presenting cells and significantly improve the efficacy of αPD-1 treatment using the B16F10 melanoma model, generating up to 20% cure rate as compared to 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+/Treg and CD8+/Treg ratios. Our work presents a novel strategy for improving cancer immunotherapy with nanotechnology.

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Section: Methodsmentioning

confidence: 99%

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

et al. 2017

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“…Subsequent steps, i.e. carbamidomethylation, alkylation and tryptic digestion, were performed exactly as described before [47,48]. …”

Section: Methodsmentioning

confidence: 99%

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Ludwig

Miroschedji

Doeppner

et al. 2018

J of Extracellular Vesicle

198

206

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Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

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“…MRM data were analysed in Skyline using an external peptide library database (http://www.peptideatlas.org) and with in‐house spectral libraries generated from previous serum LS‐MS/MS discovery experiments and from synthetic peptides. Dried peptide samples were reconstituted, and prior to analysis, serum samples were subjected to trypsin digestion using a method adapted from the FASP protocol (Wiśniewski et al ., 2009). The pre‐analytical reproducibility of serum digestions was measured and shown to be highly reproducible by MRM quantification of proteotypic peptides representing the 14 most abundant serum proteins which between them comprise > 95% total serum protein.…”

Section: Methodsmentioning

confidence: 99%

Integrating biomarkers across omic platforms: an approach to improve stratification of patients with indolent and aggressive prostate cancer

Murphy

Boyce

et al. 2018

Molecular Oncology

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Classifying indolent prostate cancer represents a significant clinical challenge. We investigated whether integrating data from different omic platforms could identify a biomarker panel with improved performance compared to individual platforms alone. DNA methylation, transcripts, protein and glycosylation biomarkers were assessed in a single cohort of patients treated by radical prostatectomy. Novel multiblock statistical data integration approaches were used to deal with missing data and modelled via stepwise multinomial logistic regression, or LASSO. After applying leave‐one‐out cross‐validation to each model, the probabilistic predictions of disease type for each individual panel were aggregated to improve prediction accuracy using all available information for a given patient. Through assessment of three performance parameters of area under the curve (AUC) values, calibration and decision curve analysis, the study identified an integrated biomarker panel which predicts disease type with a high level of accuracy, with Multi AUC value of 0.91 (0.89, 0.94) and Ordinal C‐Index (ORC) value of 0.94 (0.91, 0.96), which was significantly improved compared to the values for the clinical panel alone of 0.67 (0.62, 0.72) Multi AUC and 0.72 (0.67, 0.78) ORC. Biomarker integration across different omic platforms significantly improves prediction accuracy. We provide a novel multiplatform approach for the analysis, determination and performance assessment of novel panels which can be applied to other diseases. With further refinement and validation, this panel could form a tool to help inform appropriate treatment strategies impacting on patient outcome in early stage prostate cancer.

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Universal sample preparation method for proteome analysis

Cited by 7,010 publications

References 20 publications

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Integrating biomarkers across omic platforms: an approach to improve stratification of patients with indolent and aggressive prostate cancer

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