Protein Corona Analysis of Silver Nanoparticles ... - ACS Publications

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Protein corona analysis of silver nanoparticles links to their cellular effects Sabine Juling, Alicia Niedzwiecka, Linda Böhmert, Dajana Lichtenstein, Soeren Selve, Albert Braeuning, Andreas F. Thünemann, Eberhard Krause, and Alfonso Lampen J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00412 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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Journal of Proteome Research

Protein corona analysis of silver nanoparticles links to their cellular effects

Sabine Juling1,*, Alicia Niedzwiecka1, Linda Böhmert1, Dajana Lichtenstein1, Sören Selve2, Albert Braeuning1, Andreas F. Thünemann3, Eberhard Krause4, Alfonso Lampen1

1

BfR, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany

2

Technical University Berlin, ZE Electronmicroscopy, Straße des 17. Juni 135, 10623 Berlin, Germany

3

BAM, German Federal Institute for Materials Research and Testing, Unter den Eichen 87, 12205 Berlin, Germany

4

Leibniz Institute for Molecular Pharmacology, Robert-Roessle Str. 10, 13125 Berlin, Germany

* Corresponding Author: E-Mail address: [email protected]; Tel: +49 30 18412 3829

KEYWORDS: silver nanoparticles NM-300, protein corona,

18

O quantitative mass spectrometry, Caco-2

cells, proteomics, transcriptomics

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ABSTRACT The breadth of applications of nanoparticles and the access to food-associated consumer products containing nano-sized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic dataset was included in the analyses in order to obtain a combined multi-omics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help obtaining information about the molecular consequences of nanoparticles treatment.


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INTRODUCTION In recent years, the use of engineered nanoparticles has witnessed a strong rise in different household applications and gets access to everyday food-associated consumer products

1-2

.

Currently, nanoparticles are defined as 1) from forward (experiment 1) and reverse (experiment 2). Proteins were considered to be probably part of the silver nanoparticle corona, if the isotope ratio was higher than 2 in both experiments, i.e. if the protein was enriched in abundance following incubation of cell lysates with silver nanoparticles, as compared to control lysate without silver nanoparticles (Figure 2). This resulted in a set of 389 identified proteins (black dots inside the blue box in Figure 3A) and a detailed list of identified protein is shown in the Supplemental Table S3. Proteins with a low affinity to silver nanoparticles and heavy proteins (which form a pellet after ultracentrifugation without being specifically adherent to the nanoparticles) showed an

18

O/16O ratio of about 1 (Figure 3A). The distribution of isoelectric

points of the corona proteins demonstrates that proteins with a broad pH range are present



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in the protein corona of silver nanoparticles (Figure 3B). Most corona proteins were of comparably low molecular weight (Figure 3C).

Proteomic alterations in Caco-2 cells following exposure to silver nanoparticles Beside the identity of proteins from Caco-2 cell lysate forming the corona of silver nanoparticles, the cellular response after incubation with these nanoparticles was analyzed using a proteomic approach. This approach is partly the same for protein that were part of corona proteins and samples giving information about the cellular response. The important differences are the incubation and isolation steps as illustrated in Figure 2. To gain the cellular response, differentiated Caco-2 cells were exposed for 24 h to silver nanoparticles. Afterwards, cells were washed, harvested and proteomic changes were analyzed. Using the crossover labeling strategy depicted in Figure 3, 3518 (experiment 1) and 4590 (experiment 2) proteins were identified and quantified after nanoparticle treatment of viable Caco-2 cells with a non-cytotoxic concentration of silver nanoparticles for 24 h. A total of 1881 proteins overlapped between experiments 1 and 2. The majority of these proteins showed isotope ratios of about 1, indicating that the proteins are not regulated by the nanoparticles. Of these 1881 proteins, 54 proteins were identified as differentially regulated by silver nanoparticle treatment with the criteria of an isotope ratio >1.5 or 1.5) after matrix (30 proteins) and silver nanoparticle (54 proteins) treatment. Only one single protein, GDP-fucose proteinfucosyltransferase 1 (Q92616), overlapped and was down-regulated in both treatments (Figure 4A). For the full lists of differentially regulated proteins following incubation of Caco-2 cells with silver nanoparticles or with coating matrix with please refer to Supporting Information Table S1 and S2.

Connection of nanoparticle protein corona and nanoparticle -induced cellular changes To establish a potential correlation between the corona proteins and the cellular response to nanoparticle exposure, different approaches were used. First, a direct comparison of data sets for silver nanoparticle protein corona and protein differential regulation (proteomic response) was performed, which yielded only limited overlap at the level of individual proteins (Figure 5A). However, only a rather small number of proteins had been detected as differentially regulated by silver nanoparticles in our proteomic analysis, possibly impeding proper comparisons. In order to obtain a comprehensive picture of cellular disturbance 19 ACS Paragon Plus Environment


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following exposure of Caco-2 cells to silver nanoparticles30 a data set from a previously published toxicogenomic approach was included in subsequent analyses. This later experiment was a whole transcriptome microarray analysis of differentiated Caco-2 cells exposed for 24 h to the same type of silver nanoparticles at a concentration of 25 µg/mL and was also performed in our laboratory. These conditions of incubation ensure comparability of the datasets. The number of affected genes/transcripts (4024) was considerably higher than the number of affected proteins (54) and thus allowed for meaningful bioinformatic analysis of silver nanoparticle-induced cellular disturbance. Upon comparison with the larger data set of transcriptomic differential regulation it appeared that 139 of the 389 proteins contained in the corona of silver nanoparticles (33.4%) were also differentially regulated in their expression by silver nanoparticle treatment of Caco-2 cells at the mRNA level (Figure 5B). However, the overlap between proteome and transcriptome data showed ratios with different directions. This overlap indicated a possible direct connection of corona-forming proteins with cellular differential regulation induced by the presence of silver nanoparticles. Besides the direct correlation of protein corona and regulated proteins, nanoparticles interacting with proteins in their corona may induce or inhibit cellular metabolic and signaling pathways and thus change mRNA and protein levels of proteins that are not directly involved in the corona itself. Therefore in a next step the comparison of data sets was extended, from the level of individual proteins/transcripts to cellular functions and pathways associated with the respective proteins or RNAs. A selection was made based on the most prominent examples in the categories (cellular compartments, molecular functions and biological processes). As visualized in Figure 5C, cellular localization of corona proteins, differentially regulated proteins, and differentially regulated transcripts was comparatively analyzed using Gene Ontology (GO) demonstrating that the cellular distribution of proteins in the different data

sets

was

comparable:

all

three

data

sets

were

similarly

dominated

by

cytosolic/cytoplasmic, membrane-bound or vesicular, as well as nuclear proteins. A comparable picture of similarity was revealed when molecular functions (Figure 5D) or biological processes (Figure 5E) related to the corona proteins or differentially regulated 20 ACS Paragon Plus Environment

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proteins/transcripts were analyzed: similar in all data sets, most proteins were associated with metabolic processes and/or with cellular regulation and response to external stimuli. However, some smaller deviations between the data sets also became apparent; for example the protein corona data set contained a higher percentage of proteins involved in the binding of nucleotides (24%), nucleic acids (32%), and/or RNA (Figure 5D), as compared to the data sets for protein (19%, 19%) and transcript (14%, 23%) differential regulation. An additional evaluation of the proteomic and transcriptomic datasets revealed that considerably more proteins and transcripts were up-regulated than down-regulated regarding the different GO terms depicted in figures 5C-E (see figure S1 in the supplements). This effect was more pronounced at the protein level than at mRNA level. To better understand functional relations and pathways, bioinformatic analyses with Ingenuity Pathway Analysis (IPA) were used to obtain mechanistic information about relevant cellular processes affected by silver nanoparticles. IPA was used to simultaneously visualize the involvement of corona proteins as well as silver nanoparticle-affected proteins and transcripts in differentially regulated molecular signaling pathways. A selection of these pathways is depicted in Figure 6. All selected pathways showed that more proteins/transcripts in the respective pathways were down-regulated than up-regulated after silver nanoparticle treatment. Figure 6A shows small GTPase-dependent signaling via Rac and Rho which regulate intracellular transmission of exogenous signals and thereby influence a multitude of biological processes including apoptosis, differentiation, cytoskeletal reorganization, and membrane trafficking. Corona-forming proteins (violet border) as well as differentially regulated proteins (blue border) and transcripts (black border) dys-regulated by silver nanoparticles which are involved in Rho/Rac-dependent signaling are highlighted. In accordance with the identified molecular functions of GTP and nucleotide binding (Figure 5), the corona proteins ARHGDIA, MYl3, PI4KA, RAC1, and RHOA were identified as coronaforming proteins involved in the Rho/Rac pathway (Figure 6A). The proteins CDH17 and PAK2 which are differentially regulated after silver nanoparticle treatment of Caco-2 cells also play a role in Rho/Rac signaling. In addition, numerous genes encoding proteins 21 ACS Paragon Plus Environment


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involved in signaling through Rho and/or Rac were differentially regulated at the transcript level (Figure 6A), yielding high overall coverage of the pathway when taking into consideration protein corona and cellular differential regulation at the mRNA and protein levels. Figure 6B shows actin cytoskeleton signaling. Multiple pathways control rearrangement of the actin cytoskeleton. Again, members of the Rho family of small GTPases, including RhoA and RAC, were corona-forming proteins involved in these processes. They are activated by various classes of transmembrane receptors, for example via the integrin/FAK (focal adhesion kinase) pathway, which was well covered by the transcriptomic dataset revealing pronounced differential regulation of important key players (Figure 6B). The critical RAC downstream effector PAK which disassembles stress fibers and focal adhesion was found to be regulated by silver nanoparticles at the protein level (Figure 6A-B). PAK1 differential regulation by silver nanoparticles was also detected in the transcriptomic data set from Böhmert et al.30. Of note, a number of cytoskeletal proteins were also present in the silver nanoparticle protein corona, for example actin and myosin. A more detailed visualization of integrin-dependent signaling illustrating the differential regulation of transcripts and proteins by silver nanoparticles, as well as showing the involvement of corona proteins is given in Figure 6C. Bioinformatic analyses also predicted pronounced alterations in tight junction signaling (Figure 6D). Using the datasets from the different proteomic approaches (corona and protein differential regulation), as well as differentially regulated genes from the transcriptomics approach, the involvement of numerous corona proteins (e.g. ZO1, actin, CTNNA1, CTNNB1), as well as differentially regulated proteins or transcripts (e.g. ZO2, ZO3, OCLN, CLDN) in the formation and regulation of tight junctions is shown. Of the differentially regulated proteins/transcripts and corona proteins depicted in the figure, more (>10) are down-than up-regulated. This might be interpreted as a hint for a decrease in functionality. Moreover, down-regulation of the consecutive transcripts/proteins CLDN, ZO1 and F-Actin may indicate an overall negative impact on the subsequent target “Actin nucleation” (figure 6D). Down-regulation of specific signaling pathways by silver nanoparticles is furthermore suggested by the down-regulation of the successive 22 ACS Paragon Plus Environment

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transcripts/proteins CDC42 (ARHGEF6/7), PAK1 and LIMK in the Rho-dependent signaling cascade (figure 6A) and by the down-regulation of the consecutive transcripts FYN, SHC and SOS in the integrin-associated signaling cascade (figure 6C). Thus, silver nanoparticles seem to negatively affect distinct signaling pathways by similarly down-regulating more than one member of the respective signaling cascade either at the transcript or at the protein level.

DISCUSSION In a biological environment nanoparticles interact with biopolymers like proteins to form complex surfaces, impacting the interaction between nanoparticles and cellular structures. Thus, the identity of the proteins that bind to silver nanoparticles, also termed protein corona, could determine effects of nanoparticles on cellular functions. The initial protein corona of the particles formed in FCS-containing medium primarily consists of albumin20-21, 40-41 and might influence cellular particle uptake. However, out of methodological considerations and especially because an exchange of albumin for cellular proteins is expected following particle uptake into a cell, this primary corona was not taken into consideration in the current experiment. Instead, our corona formation experiment started with formation of the protein corona inside the cell by incubating nanoparticles with cell lysates. Even though this model might not cover all possible effects of the primary, extracellular FCS-derived corona, the present data show its usefulness in unraveling the molecular connection of the intracellular protein corona with effects at the proteome and/or transcriptome levels. To gain insight in these correlations between nanoparticle corona protein identification and cellular responses we investigated the molecular and cellular mechanisms of silver nanoparticles in differentiated Caco-2 cells, a model cell line for the intestine, with the two major focuses on the formation of protein corona as well as the resulting proteomic effects after silver nanoparticle treatment. Nanoparticle sedimentation, cellular uptake, ion release and the time needed for proteomic responses (e.g. via altered protein degradation or via 23 ACS Paragon Plus Environment


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RNA and protein synthesis) are considered to be rather slow and continuously occurring processes, which are therefore expected to lead to rather continuous proteomic or transcriptomic responses which might change gradually over time. Therefore, we choose a 24h nanoparticle incubation for the investigation of proteomic effects. By contrast, coronaforming proteins were isolated from cell lysates after 2h of incubation. This was done based on initial studies on time-dependent protein corona formation in cell lysate which indicated no substantial differences between the protein coronas formed after 1h, 2h, or 24h of nanoparticle incubation in cell lysate. Therefore, we used a quantitative mass spectrometry- based proteomic approach to identify corona-forming and differentially regulated proteins (proteome response). In addition, we combined our results with our previous transcriptomics analysis30 to get a more comprehensive view on the context of altered biological functions. It is remarkable that strikingly different numbers of deregulated RNAs and proteins have been disvocered following identical cell treatment. One underlying reason might be that, based on differences in methodology, the number of detected and identified proteins is generally below the number of transcripts analyzed in a microarray experiment. In addition, changes at the mRNA level are not always reflected by concomitant changes at the protein level and the dynamic range of deregulation (x-fold up- or down-regulation) is generally considered to be remarkably broader at the mRNA level, as compared to proteins. We identified 389 coronaforming and 54 differentially regulated proteins after silver nanoparticle treatment which fits well with other proteomics studies, the latter of which showed differential regulation of >10 and 3) showed the absence of cytotoxicity at the silver nanoparticle concentration used in the experiment. The coating material (right bar) did not exert cytotoxicity at a concentration corresponding to the amount of coating material present in cell culture during incubation with 100 µg/mL silver nanoparticles. (D) Representative transmission electron microscopy (TEM) image with electron dispersive X-ray spectroscopy (EDX) shows the uptake of silver nanoparticles in Caco-2 cells.



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Figure 2:

Figure 2: Experimental workflow. Silver nanoparticle treatment of differentiated Caco-2 cells was used for analysis of de-regulated proteins. Characterization of intracellular protein corona was performed via ultracentrifugation of Caco-2 cell lysate following by incubation with silver nanoparticles. Protein identification and relative quantification was achieved using 1D LC-ESI-MS/MS and stable isotope labeling via 18O-water.


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Figure 3:

Figure 3: Mass-spectrometric identification of corona proteins. (A) Plot of isotope ratios (log2 fold) of identified proteins determined in two independent experiments (experiment 1 and 2) performed in a crossover design (i.e., forward and reverse stable isotope labeling with and

16

O for each experiment). The forward experiment shows the results of

isotope labeling with nanoparticles) and

16

18

18

O

the stable

O for the corona forming proteins (enriched proteins which bind to the

O for the protein background (ultracentrifuged cells without nanoparticle

treatment) and the reverse labeling, which means corona forming proteins are labeled with 16

O and the untreated cells are labeled with heavy water H218O. Every dot represents one

protein, which was found in both independent experiments (1 and2). The blue box shows the protein corona proteins (389 proteins) with log2 ratio > 2. A selection of corona proteins involved in relevant molecular signaling pathways as depicted in Figure 6 is indicated. (B) The distribution of isoelectric points (pI) shows that proteins with a broad pI range form the corona. (C) Proteins with a low molecular weight were predominantly forming the protein corona of silver nanoparticles. For a detailed list of identified corona proteins, please refer to Supplemental Table S3.



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Figure 4:

Figure 4: Proteomic analysis of Caco-2 cells following incubation with silver nanoparticles. (A) Overlap among proteins regulated by silver nanoparticles and by the particle coating matrix. 30 proteins were differentially regulated by matrix (4 up and 26 down) and 54 by nanoparticle treatment (42 up and 12 down) with one overlapping (down-regulated) protein. (B) Plot of isotope ratios (log2 fold) of identified proteins after 24 h nanoparticle treatment (54) determined in two independent experiments performed in a crossover manner (forward and reverse stabile isotope labeling). Every dot represents one protein. 42 up and 12 down regulated proteins are located inside the blue boxes. A selection of corona proteins involved in relevant molecular signaling pathways as depicted in Figure 6 is indicated. (C) Plot of isotope ratios of identified de-regulated proteins after matrix treatment (30). 26 up and 4 down-regulated proteins are located inside the blue boxes.


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Figure 5:

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Figure 5: Bioinformatic analysis of the silver nanoparticle protein corona (blue color) as well as de-regulated proteins (red) and transcripts (gray)

in Caco-2 cells following exposure to silver nanoparticles. The transcriptomic data set was taken from a previous study conducted under comparable conditions30. (A) Visualization of overlap of the protein corona and protein deregulation data sets. (B) Visualization of overlap of the protein corona, protein deregulation, and transcript deregulation data sets. Please note that the two protein data sets had to be converted into sets of corresponding transcripts to allow for the comparison with the transcriptomic data. Data sets were separately subjected to gene ontology (GO) analysis. The diagrams show the percentage (i.e. normalized to the total number of identified differentially regulated proteins/ transcripts) of data points from each data set for the GO terms for cellular localization (C) biological process (D), and molecular function (E).

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Figure 6:



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Figure 6: Bioinformatic analysis of molecular pathways in Caco-2 cells affected by silver nanoparticle treatment and also involving silver nanoparticle corona proteins. Schematic representations of Rho-dependent Signaling (A), actin cytoskeleton signaling (B), integrin signaling (C), and tight junction signaling (D) are shown. Pathway charts were adapted using Ingenuity Pathway Analysis (IPA) software. De-regulated proteins are indicated by blue border with filling in red or green color for up- and down-regulation, respectively. Differential regulated transcripts are shown by black border, also with filling in red or green color for upand down-regulation. Corona proteins are depicted with a violet border with no filling. Uncolored elements within the pathways are predicted linking proteins not affected by silver nanoparticle treatment and not present in the silver nanoparticle protein corona.

Table 1: Set of the 10 top (A) up- and (B) down-regulated proteins after silver nanoparticles tratment in the proteomics approach.

A

Isotop e Ratio (FF)

Isotope Ratio (REV)

Q6UWP8

16.87

114.53

T22D2

O75157

5.49

2.45

Band 4.1-like protein 2

E9PHY5

E9PHY5

3.47

3.43

Protein PRRC2A

PRC2A

P48634

2.94

2.01

Tight junction protein Z

ZO2

Q9UDY2

2.68

1.89

Carcinoembryonic antigen-related cell adhesion molecule 1

CEAM1

P13688

2.38

3.02

Cadherin-17

CAD17

Q12864

2.3

5.52

Nucleolar RNA helicase 2

DDX21

Q9NR30

2.12

29.05

Sulfotransferase family cytosolic 1B member 1

ST1B1

O43704

1.99

76.51

Niban-like protein 1

NIBL1

Q96TA1

1.86

2.39

B

Protein ID

Accession

HYEP

P07099

Protein ID

Accession

Suprabasin

SBSN

TSC22 domain family protein 2

Description

Description Epoxide hydrolase 1

Isotope Ratio (FF)

Isotope Ratio (REV)

0.54

0



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NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial

NDUFS7

O75251

0.67

0.03

Serum paraoxonase/lactonase 3

PON3

Q15166

0.58

0.06

40S ribosomal protein S15a

RS15A

P62244

0.37

0.06

NADH dehydrogenase (Ubiquinone) 1 alpha subcomplex, 13

GRIM-19

Q9P0J0

0.27

0.07

Liver carboxylesterase 1

EST1

P23141

0.61

0.15

MpV17 transgene, murine homolog, glomerulosclerosis, isoform CRA_f

B5MC53

B5MC53

0.58

0.21

N-acetylated-alpha-linked acidic dipeptidase 2

J3KNJ3

J3KNJ3

0.4

0.32

Cytochrome b5

CYB5

P00167

0.44

0.46

Serine/threonine-protein kinase PAK 2

PAK2

Q13177

0.61

0.55


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Protein Corona Analysis of Silver Nanoparticles ... - ACS Publications

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