A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro

Figures & data

Figure 1. Experimental design. A combination of 2D-gel based proteomic and MS-based metabolomic approaches was used to analyze the differentially expressed proteome and metabolites of the cytoplasmic compartment of Caco-2 cells exposed to 5 or 30 nm AuNPs (300 μM) for 72 h. Data obtained were interpreted using a combination of bioinformatics tools for a combined omics approach.

Figure 2. Proteomic analysis of the cytoplasmic extract of Caco-2 cells exposed to AuNPs. Representative two-dimensional gel protein maps of cytoplasmic fractions of (A) untreated (Ctrl), (B) treated with 5 nm AuNPs, and (C) treated with 30 nm AuNPs cells for 72 h. (D) The Venn diagram is showing the distribution of differentially expressed proteins: 5 nm AuNPs versus Ctrl (red circle, 36 proteins), 30 nm AuNPs versus (blue circle, 33 proteins) or 5 versus 30 nm AuNPs (green circle, 23 proteins). Number inside overlapping region of two circles refers to the spots common to different groups (For color figure refer to the online version.)

Figure 3. Biological functions altered by AuNPs exposure. Bar charts grouped by biological function representing the differentially expressed proteins and metabolites, after 72 h of treatment with AuNPs. (A) Represents the de-regulated proteins by 5 nm or 30 nm AuNPs and (B) the de-regulated metabolites following exposure to 5 and 30 nm AuNPs.

Table 1. List of de-regulated proteins identified in individual 2D gel spots of Caco-2 cytoplasmic extracts.

Each protein spot has been assigned a UniProt accession number, the protein symbol, the protein coverage, the number of identified peptides and amino acids, the molecular mass, the calculated pI, and a probability score. Proteins have been classified according to their main function based on UniProtKB/Swiss-Prot and Gene Ontology (GO) (UniProt, Washington, DC). Quantitative changes after AuNPs treatment (5 nm AuNPs versus Ctrl and 30 nm AuNPs versus Ctrl) are reported (p value and fold change) for individual proteins. Log 2 = 0.58, thus the range was set from −0.58 to 0.58 and color-coded. Light green for < 0.58, and dark red for < 0.58. The values in between are shown as color gradients. (For color table refer to the online version.)

Table 2. List of de-regulated tentatively annotated metabolites in the cell extract.

Metabolites have been assigned HMBD tags and have been grouped according to their biological functions. Quantitative changes after AuNPs treatment (5 nm AuNPs versus Ctrl and 30 nm AuNPs versus Ctrl) are reported (log 2-fold change) for individual metabolites. Log 2 = 0.58, thus the range was set from –0.58 to 0.58 and color-coded. Green for < –0.58, black ▪ for 0 and red for > 0.58. The values in between are shown as color gradients. (For color table refer to the online version.)

Figure 4. Molecular networks. De-regulated molecular networks in response to 5 or 30 nm AuNPs exposure in Caco-2 cells (300 μM, 72 h). The networks are obtained by analyzing the differentially expressed proteins and metabolites (listed in Tables 2 and 3) using Ingenuity IPA. Identified de-regulated proteins and metabolites involved in the network are highlighted in bold. The color indicate the de-regulation (red

: up-regulated, green

: down-regulated). (A) Cellular compromise (degeneration); (B) Small molecule biochemistry; (C) cell morphology; (D) cellular assembly and organization; (E) cellular assembly and organization according to Table 3. (For color figure refer to the online version.)

Table 3. Identified molecular networks using Ingenuity IPA.

The table reports the most significant molecular networks in response to 5 and 30 nm AuNPs treatments, by analyzing the differentially expressed proteins (listed in Table 1) and metabolites (listed in Table 2) from the cells. The network number, the list of all proteins and metabolites involved in the network, the number of molecules overlapping between our data set, and the network and the top functions related to the network are shown. Focus molecules are indicated in bold and de-regulation is indicated with a colored arrow (dark red : up-regulated, light green : down-regulated). (For color table refer to the online version.)