Fig 1.
Experimental setup to study proteome-wide biological responses to non-ionizing EMFs using semi-quantitative mass spectrometry.
Triple-state (triplex) SILAC proteomics with reverse metabolic labeling of human fibroblasts (VH10), human osteosarcomas (U2OS) and mouse embryonic stem cells (IB10) exposed to different EMFs with extremely low (ELF) or radio frequencies (UMTS or WiFi). Cells were cultured in media containing “Light” (Arg-0/Lys-0), “Medium” (Arg-6/Lys-4) and “Heavy” (Arg-10/Lys-8) stable isotopes. Cultures were sham (denoted as L0, M0 and H0) or exposed (denoted as L1, M1 and H1) to EMFs. Two independent LC-MS experiments of mixtures of cell extracts were performed: two sham and one exposed extract in the mixture (L0+M0+H1, indicated as replicate 1), and two exposed extracts with one sham extract in the mixture (L1+M1+H0, indicated as replicate 2). Note that the L and M samples (duplicates) were treated equally in both experiments and could therefore be used as internal controls to quantify the experimental variation due to cell culturing, metabolic labeling and/or preparing the samples for mass spectrometry analysis. Further downstream bioinformatics analyses involved peptide/protein identification and quantitation, and the detection of differentially regulated proteins and perturbed cellular processes or pathways.
Fig 2.
Semi-quantitative proteomics data management and analysis.
The SILAC-based mass spectrometry data from EMF exposed mammalian cells were analyzed by the MaxQuant/Andromeda software. The resulting peptide/protein identifications and quantifications were uploaded to the PIQMIe proteomics server, which integrated the EMF exposure data with protein information from UniProtKB and made the databases available for user-driven queries and statistical analyses.
Table 1.
Summary of mass spectrometry-based protein identifications in human and mouse proteomes exposed to EMFs.
Table 2.
Two- and three-way set comparisons of protein identifications from the EMF exposure experiments.
Table 3.
Summary of (non-redundant) protein identifications/quantifications in human (VH10 and U2OS) and mouse (IB10) cells exposed to EMFs.
Fig 3.
Correlation analysis of reciprocal SILAC protein quantifications in three mammalian cell lines exposed to three different EMFs.
(A-C) Three scatterplots based on simulated SILAC protein ratios from reverse labeling experiments—with treated (H1 or L1) versus sham (L0 or H0) samples on both axes—illustrate three possible scenarios in which the scatter depends on the effect of a treatment (e.g. EMF exposure) versus SILAC reverse labeling: (A) the scatter is in the direction of the treatment, as indicated by a positive Pearson's correlation coefficient (r); (B) the scatter does not have an identifiable trend, as indicated by a value of r close to zero; (C) the scatter is in the direction of the SILAC reverse labeling, as indicated by a negative value of r. Each scatterplot is divided into four quadrants (I-IV): proteins with inconsistent SILAC reciprocal ratios are located in the I and III quadrants whereas proteins with consistent up- and down-regulation upon treatment are located in the II and IV quadrants, respectively. (D) The dot plot summarizes quantitative data from human (U2OS and VH10) and mouse (IB10) cell lines exposed to ELF, UMTS and WiFi (the individual scatterplots are shown in S2–S4 Figs); the r estimates including the error bars (95% confidence interval) are based on SILAC protein ratios from the H-M (in blue) and the H-L (in red) reverse labeling experiments. Note that the estimated r values are negative in all EMF exposures and hence correspond to the third scenario illustrated by the scatterplot (C).
Fig 4.
Variability of SILAC protein ratios in human U2OS cells upon ELF, UMTS and WiFi exposures.
Median absolute deviation (MAD) is used as a robust measure of variability in SILAC protein ratios. Note that unfiltered refers to an unfiltered SILAC data set containing all quantitated protein groups; (in)consistent refers to a filtered SILAC data set of protein groups with (in)consistent ratios in both reverse labeling experiments, this fold-change filtering procedure is only possible because of the duplex SILAC design; composite refers to a filtered SILAC data set of protein groups with greater 'treated' ratios than 'control' ratios (in total there are four 'treated' ratios from exposed versus sham samples, and two 'control' ratios from exposed versus exposed samples and sham versus sham samples), this (composite) fold-change filtering procedure requires triplex SILAC design. The results for the human VH10 and mouse IB10 cells are shown in S5 Fig.
Table 4.
Differentially regulated proteins detected upon EMFs in human (VH10 and U2OS) and mouse (IB10) cells using an array of statistical methods.
Fig 5.
Quantitative immunoblot analysis of MLH1 expression in the VH10 cell line upon ELF exposure.
(A) Immunoblot of control cell line HEK293T-Lα [25] in which expression of DNA mismatch repair protein MLH1 and its binding partner PMS2 is regulated by doxycycline. The presence (+) and absence (-) of doxycycline is indicated. (B) Immunoblot of extracts from untreated (SHAM) and exposed (ON) VH10 cells labeled with different isotopes (L, M and H) and used for semi-quantitative mass-spectrometry analysis. Proteins were visualized using antibodies against MLH1, its binding partner PMS2, and α-tubulin as loading control. (C) Relative MLH1 expression levels (mean ± SD) in extracts from untreated and exposed cells as determined from 6 immunoblot replicas of the MS samples. (D) Tabulated ratios for relative MLH1 expression levels in differentially labeled VH10 cells as determined by mass spectrometry (MS) and immunoblot (IB) analyses. (E) Immunoblot of VH10 cells, lysed using either urea or SDS, obtained from an independent exposure. (F) Relative MLH1 expression levels (mean ± SD) in extracts from an independent exposure as determined from 9 immunoblot replicas. (G) Tabulated ratios for relative MLH1 expression levels based on immunoblot analysis of extracts from the independent exposure.
Fig 6.
Network of differentially regulated mammalian proteins associated (annotated) with biological processes.
In this network, the proteins and biological processes (referred by the Gene Ontology (GO) terms) are indicated by oval- and box-shaped nodes, respectively; human proteins (gray) and mouse (white) proteins detected as differentially regulated upon ELF (solid), UMTS (dashed) and WiFi (dot-dashed) exposures; protein-to-GO term associations are indicated by edges labeled with GO evidence codes: inferred from direct assay (IDA); inferred from mutant phenotype (IMP); inferred from genetic interaction (IGI); traceable author statement (TAS); non-traceable author statement (NAS); inferred from sequence orthology (ISO); inferred from electronic annotation (IEA). For additional details on the proteins refer to S5 Table.