Analytical constraints for the analysis of human cell line secretomes by shotgun proteomics

Malard, Véronique; Chardan, Laetitia; Roussi, Stamatiki; Darolles, Carine; Sage, Nicole; Gaillard, Jean‐Charles; Armengaud, Jean

doi:10.1016/j.jprot.2011.10.025

Cited by 17 publications

(10 citation statements)

References 48 publications

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“…Even if cell viability is very high (higher than 90%), these intracellular proteins are detected due to the high sensitivity of the mass spectrometer used here. Definitively, the number of secreted proteins observed for ZF4 cells in this condition is low compared to previous studies carried out with human cell lines [47]. This may be primarily due to the short incubation time, i.e.…”

Section: Resultscontrasting

confidence: 61%

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Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells

et al. 2014

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The term “bystander effect” is used to describe an effect in which cells that have not been exposed to radiation are affected by irradiated cells though various intracellular signaling mechanisms. In this study we analyzed the kinetics and mechanisms of bystander effect and radioadaptation in embryonic zebrafish cells (ZF4) exposed to chronic low dose of gamma rays. ZF4 cells were irradiated for 4 hours with total doses of gamma irradiation ranging from 0.01–0.1 Gy. In two experimental conditions, the transfer of irradiated cells or culture medium from irradiated cells results in the occurrence of DNA double strand breaks in non-irradiated cells (assessed by the number of γ-H2AX foci) that are repaired at 24 hours post-irradiation whatever the dose. At low total irradiation doses the bystander effect observed does not affect DNA repair mechanisms in targeted and bystander cells. An increase in global methylation of ZF4 cells was observed in irradiated cells and bystander cells compared to control cells. We observed that pre-irradiated cells which are then irradiated for a second time with the same doses contained significantly less γ-H2AX foci than in 24 h gamma-irradiated control cells. We also showed that bystander cells that have been in contact with the pre-irradiated cells and then irradiated alone present less γ-H2AX foci compared to the control cells. This radioadaptation effect is significantly more pronounced at the highest doses. To determine the factors involved in the early events of the bystander effect, we performed an extensive comparative proteomic study of the ZF4 secretomes upon irradiation. In the experimental conditions assayed here, we showed that the early events of bystander effect are probably not due to the secretion of specific proteins neither the oxidation of these secreted proteins. These results suggest that early bystander effect may be due probably to a combination of multiple factors.

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Section: Resultscontrasting

confidence: 61%

“…Nano-liquid chromatography-tandem MS (LC-MS/MS) experiments were performed using a LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex-LC Packings) in similar conditions as those previously described [47]. A volume of 10 μL of peptide mixture was loaded per sample.…”

Section: Methodsmentioning

confidence: 99%

Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells

et al. 2014

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“…Recently, Berredo-Pinho et al reported the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau and identified 101 different proteins, of which 53 were thought to be secreted proteins (14). Although proteome-wide studies performed on CFPs of BCG are still limited because of their frequently low concentrations, recent developments in Fourier transform MS with high resolution and accuracy at both the MS and MS/MS levels might substantially promote comprehensive investigations of secreted protein profiling (15). Moreover, to reduce the complexity of the extracted CFPs, a onedimensional gel electrophoresis separation outperformed previous strategies with respect to the number of identified proteins, reproducibility, and throughput (9).…”

Section: Tuberculosis (Tb)mentioning

confidence: 99%

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

Zheng

Ren

Wei

et al. 2013

Molecular & Cellular Proteomics

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Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%-80%) in different trials, Mycobacterium bovis bacillus Calmette-Gué rin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on the mycobacterial secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture superna-

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“…Regarding the analysis of proteins in the culture supernatants, comprehensive exoproteomics is tedious to perform because of the highly diluted nature of secreted proteins present in biological fluids or culture media [ 30 ]. Consequently, we had to concentrate the supernatants by precipitation prior to their analysis with a sensitive tandem mass spectrometer.…”

Section: Discussionmentioning

confidence: 99%

High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field

et al. 2015

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BackgroundThe toxicity of manufactured fumed silica nanoparticles (NPs) remains poorly investigated compared to that of crystalline silica NPs, which have been associated with lung diseases after inhalation. Amorphous silica NPs are a raw material for manufactured nanocomposites, such as cosmetics, foods, and drugs, raising concerns about their potential toxicity.ResultsThe size of the NPs was determined by dynamic light scattering and their shape was visualized by atomic force microscopy (10 ± 4 nm). The pertinent toxicological concentration and dynamic ranges were determined using viability tests and cellular impedance. We combined transcriptomics and proteomics to assess the cellular and molecular effects of fumed silica in A549 human alveolar epithelial cells. The “no observed transcriptomic adverse effect level” (NOTEL) was set to 1.0 μg/cm2, and the “lowest observed adverse transcriptional effect level” (LOTEL) was set at 1.5 μg/cm2. We carried out genome-wide expression profiles with microarrays and identified, by shotgun proteomics, the exoproteome changes in lung cells after exposure to NP doses (0.1, 1.0, 1.5, 3.0, and 6.0 μg/cm2) at two time points (24 h and 72 h). The data revealed a hierarchical, dose-dependent cellular response to silica NPs. At 1.5 μg/cm2, the Rho signaling cascade, actin cytoskeleton remodeling, and clathrin-mediated endocytosis were induced. At 3.0 μg/cm2, many inflammatory mediators were upregulated and the coagulation system pathway was triggered. Lastly, at 6.0 μg/cm2, oxidative stress was initiated. The proteins identified in the extracellular compartment were consistent with these findings.ConclusionsThe alliance of two high-throughput technologies allowed the quantitative assessment of the cellular effects and molecular consequences of exposure of lung cells to low doses of NPs. These results were obtained using a pathway-driven analysis instead of isolated genes. As in photography, toxicogenomics allows, at the same time, the visualization of a wide spectrum of biological responses and a “zoom in” to the details with a great depth of field. This study illustrates how such an approach based on human cell culture models is a valuable predictive screening tool to evaluate the toxicity of many potentially harmful emerging substances, alone or in mixtures, in the framework of future regulatory reinforcements.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1521-5) contains supplementary material, which is available to authorized users.

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Analytical constraints for the analysis of human cell line secretomes by shotgun proteomics

Cited by 17 publications

References 48 publications

Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells

Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field

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