Dataset Information

Automated method for enrichment of newly synthesized proteins based on AHA labeling

ABSTRACT: In this study, a novel high throughput method was successfully developed for the automated enrichment of newly synthesized proteins (NSPs) from complex environments, like cell culture media supplemented with fetal bovine serum (FBS). The proposed automated method allows for the robust and effective simultaneous enrichment of dozen of samples in a period of 3 hours. The enrichment method was comprehensively evaluated and validated, and was applied to the mass spectrometry (MS) analysis of the FBS-rich secretome of melanoma A375 cells treated with the immune cytokines interferon alpha (IFN-α) and gamma (IFN-γ).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanocyte, Cell Culture

DISEASE(S): Melanoma

SUBMITTER: David Vargas Diaz

LAB HEAD: Maarten Altelaar

PROVIDER: PXD025255 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

Automated High-Throughput Method for the Fast, Robust, and Reproducible Enrichment of Newly Synthesized Proteins.

Vargas-Diaz David D Altelaar Maarten M

Journal of proteome research 20211203 1

A high-throughput method was developed for the automated enrichment of newly synthesized proteins (NSPs), which are labeled metabolically by substituting methionine with the "click-able" analogue azidohomoalanine (AHA). A suitable conjugate containing a dibenzocyclooctyne (DBCO) group allows the specific selection of NSPs by a fast 1 h click chemistry-based reaction with AHA. Through an automated pipetting platform, the samples are loaded into streptavidin cartridges for the selective binding of ...[more]

PMID: 34860524

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Project description:The immune mechanisms that control resistance vs. susceptibility to mycobacterial infection in humans were investigated by studying leprosy skin lesions, the site where the battle between the host and the pathogen is joined. Using an integrative genomics approach, we found an inverse correlation between of IFN-beta and IFN-gamma gene expression programs at the site of disease. The Type II IFN, IFN-gamma and its downstream vitamin D-dependent antimicrobial genes were preferentially expressed in the lesions from patients with the self-healing tuberculoid form of the disease and mediated antimicrobial activity against the pathogen, Mycobacterium leprae in vitro. In contrast, the Type I IFN, IFN-beta and its downstream genes, including IL-27 and IL-10, were induced in monocytes by M. leprae in vitro, and were preferentially expressed in the lesions of disseminated and progressive lepromatous form. The IFN-gamma induced macrophage antimicrobial response was inhibited by IFN-beta/IL-10, by a mechanism involving blocking the generation of bioactive 1,25-dihyroxy vitamin D as well as inhibiting induction of antimicrobial peptides cathelicidin and DEFB4. The ability of IFN-M-oM-^AM-" to inhibit the IFN-gamma induced vitamin D pathway including antimicrobial activity was reversed by neutralization of IL-10, suggesting a possible target for therapeutic intervention. Finally, a common IFN-beta and IL-10 gene signature was identified in both the skin lesions of leprosy patients and in the peripheral blood of active tuberculosis patients. Together these data suggest that the ability of IFN-beta to downregulate protective IFN-gamma responses provides one general mechanism by which some bacterial pathogens of humans evade protective host responses and contribute to pathogenesis. Peripheral blood monuclear cells derived through Ficoll-Hypaque from the blood of healthy human donors (n=4). Cells adhered to tissue culture treated 6-well plates for 2 hours in 1% Fetal Bovine Serum (FBS) RPMI and then stimulated by IL-10 (R&D Systems) 10ng/ml or media alone in 10% FBS RPMI for 24 hours at 37M-BM-0C, 5% CO2. After stimulation, cells harvested and monocytes isolated through CD14 positive selection (Miltenyi Biotec). RNA from purified monocytes extracted by Trizol and purified by Qiagen RNeasy Kit. RNA probe and microarray performed by UCLA Clinical Microarray Core using Ambion labeling kit and Affymetrix Human U133 Plus 2.0 array.

Dataset Information

Automated method for enrichment of newly synthesized proteins based on AHA labeling

Publications

Automated High-Throughput Method for the Fast, Robust, and Reproducible Enrichment of Newly Synthesized Proteins.

Similar Datasets

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