Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively.

Proteomic techniques using tandem mass spectrometry (MS) and bioinformatics have been developed rapidly over the past two decades. Whereas proteomics studies previously relied on gel electrophoresis [13], gel-free shotgun proteomics techniques are now widely used to screen target proteins [14]. However, tandem MS remains critical to the evaluation and verification of biomarkers after identification with genomics and quantitative proteomic comparison of normal and abnormal specimens [15,16]. Multidimensional gel electrophoresis or multidimensional liquid chromatography (MDLC) techniques can limit numbers of candidate biomarkers [17][18][19][20]. However, numbers of identified nonspecific biomarker candidates often hamper evaluation and verification. As alternatives, target protein screening can be achieved using activity-based chemical probes that detect proteomic profiles according to carbon electrophiles [21] or activity-based proteomics that generate serine hydrolase enzymes [22]. Similar methods involve the design of probes using click chemistry to connect proteins and carriers [21,23], the use of chemiluminescent bioprobes [24], and Au nanoparticles linked with synthetic DNA to detect estrogen receptors [25].
In previous studies, phthalic acid was observed as a secondary metabolite from phthalate derivation, which is observed in dialysis patients [2]. In addition, the phthalic acid metabolites of DEHP and MEHP were described [26]. In the present study, we used phthalic acid as a phthalate precursor to synthesize esterified phthalic acid chemical probes and detect protein-protein interactions. Previously, we developed chemical probes that generate phthalic acid or nicotinic acid using 3-aminopropyl triethoxyslane (APTES) linkers on silicon dioxide particles [27,28]. In our previous study, BBP promoted progression of a breast cancer cell line by inducing lymphoid enhancer factor 1 [29]. For the present study, we used chemical probes to characterize phthalic acid-binding proteins in MCF-7 and MDA-MB-231 cells. Subsequently, quantitative proteomics analyses identified 22 binding proteins that were common to both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. Finally, ATP synthase subunit beta, heat shock protein HSP 90-beta, and heat shock cognate 71-kDa protein-linked proteasome protein were identified as exclusive MCF-7 proteins, and connected ribosomal correlated proteins were identified as specific to MDA-MB-231 cells.

Identification and Quantitation of Phthalic Acid-Binding Proteins Using Proteomics
Chemical probes were individually incubated with MCF-7 and MDA-MB-231 cell lysates, and phthalic acid-bound proteins were identified using LC-MS/MS ( Figure 1). After reduction by DL-dithiothreitol (DTT) and alkylation by iodoacetamide (IAM), related proteins were extracted and eluted using 1% sodium dodecyl sulfate (SDS). SDS was then removed by trichloroacetic acid (TCA) precipitation and proteins were subjected to tryptic digestion. Tryptic peptides bound to probes 1 and 2 were labeled with formaldehyde-H2 and formaldehyde-D2, respectively. Labeled samples were acidized using 10% trifluoroacetic acid (TFA) and were then desalted using a C18 desalting cartridge. Subsequently, eluted samples containing peptide mixtures were examined using LC-MS/MS, and raw data were generated using Raw2MSM (version 1.10_2007.06.14) [32] for protein characterization and Mascot Distiller (version 2.4.2.0 (64 bits)) for protein quantitation.

Phthalic Acid-Binding Proteins in MCF-7 and MDA-MB-231 Cell Lines
Protein quantitation using Mascot Distiller and manual statistics showed that ATP synthase subunit beta, the heat shock protein family, and elongation factor 1-alpha 1 bound phthalic acid chemical probes, and related proteins were found in MCF-7 and MDA-MB-231 cell lines. Furthermore, phthalic acid chemical probes were bound to numerous proteasome-related and energy-correlated proteins such as NAD(P)H dehydrogenase, UDP-glucose 6-dehydrogenase, and fatty acid synthase in MCF-7 cells. Among the correlated proteins (Table 1), the phthalic acid probes detected ribosomal proteins such as 60S acidic ribosomal proteins and 40S ribosomal proteins, ras-related proteins, and heat shock proteins including 60-kDa heat shock protein and heat shock 70-kDa protein 6.

Identification of Related Proteins Using Phthalic Acid Chemical Probes
Proteins were identified after conversion of raw MS data using Raw2MSM software with the Mascot search engine. ATP synthase subunit beta, heat shock protein HSP 90-beta, and heat shock cognate 71-kDa protein were characterized according to MS/MS patterns, and the peptide VALTGLTVAEYFR of the ATP synthase subunit beta protein showed b-and y-ion patterns (Figure 2A). Mean ATP synthase subunit beta protein quantities were 4.1-and 9.5-fold in MCF-7 and MDA-MB-231 cells (n = 3), respectively. Subsequent isotope labeling of VALTGLTVAEYFR using formaldehyde-D2 and formaldehyde-H2 ( Figure 2B) gave an m/z of 734.46 with a charge 2 + and an m/z of 736.47, respectively. The heat shock cognate 71-kDa protein was also characterized using tandem MS, and the representative peptide DAGTIAGLNVLR was identified with a charge of 2 + . MS/MS spectra ( Figure 3A) and quantitative data showed 4.2-and 4.6-fold increases in this peptide in MCF-7 and MDA-MB-231 cells (n = 3), respectively with m/z values of 616.40 (D-labeled peptide) and 614.39 (H-labeled peptide; Figure 3B). Finally, the related heat shock protein HSP 90-beta ( Figure 4A) and the peptide GVVDSEDLDLNISR were identified with a charge of 2 + , along with a D-labeled peptide m/z of 773.47 and a H-labeled peptide m/z of 771.46 ( Figure 4B). Finally, quantitative ratios showed 9.1-and 15.0-fold increases in MDA-MB-231 and MCF-7 cell lines, respectively.

Relationships between Protein-Protein Interactions in MCF-7 and MDA-MB-231 Cells
Proteins that bind phthalic acid chemical probes were identified and relationships between these were characterized by organized protein-protein interactions using STRING software. Phthalic probes demonstrated arrangements of proteasome subunit proteins (red circle), and interactions with ATP synthase subunit beta (ATP5B) and heat shock 70-kDa protein 1A/1B (HSPA8) in MCF-7 cells ( Figure 5). Furthermore, HSPA8 proteins interacted with heat shock protein HSP 90-beta (HSP90AB1), heat shock protein HSP 90-alpha (HSP90AA1), and nucleophosmin protein (NPM1). In MDA-MB-231 cells, protein-protein interactions ( Figure 6) were found between HSPA8 and HSP90AB1, NPM1, ATP5B, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and elongation factor 1-alpha 1 (EEF1A1). Moreover, among related proteins, NPM1, GAPDH, HSP90 AB1, and ATP5B interacted with EEF1A1, which was correlated with a group of ribosomal proteins (red circle). Figure 5. STRING protein-protein interactions between phthalic acid-binding proteins in MCF-7 cells. Red circles containing proteasome subunit proteins and the three red arrows indicate heat shock cognate 71-kDa protein, heat shock protein HSP 90-beta, and ATP synthase subunit beta. Figure 6. Schematic of the relationship between phthalic acid-binding proteins in MDA-MB-231 cells. High fold ratios were observed in triplicate experiments; the red circle includes numerous ribosomal proteins and the three red arrows indicate heat shock cognate 71-kDa protein, heat shock protein HSP 90-beta, and ATP synthase subunit beta. (Miamisburg, OH, USA). Trypsin was purchased from Promega (Madison, WI, USA). Phthalic acid, APTES, potassium bromide (FTIR grade), and N-hydroxysuccinimide (NHS) were purchased from Alfa Aesar (Heysham, UK). Deionized water was obtained with a resistance of 18.2 MΩ using a Millipore water system (Millipore, Bedford, MA, USA).

Synthesis and Characterization of Phthalic Acid Chemical Probes
The chemical probes were synthesized and characterized according to previous studies [27,28]. Briefly, 200 mg of silicon dioxide (SiO2, 400 mesh, approximately 40 μm; Acros Organics, Geel, Belgium) was activated using 0.5 M HCl and 0.5 M NaOH, then washed and dried with distilled water and ethanol to remove and evaporate HCl and NaOH. Surface silanization of SiO2 was performed by reacting with APTES (5% in ethanol), and the SiO2 was washed two times with 1 mL of ethanol and was baked overnight in an oven at 50 °C. Subsequently, 13 mg of EDC and 5 mg of NHS were added in 1 mL of deionized water to react with 10 mg of phthalic acid. After activation by EDC/NHS, the phthalic acid was conjugated to SiO2 via the amino groups of APTES. Functional groups of APTES-modified SiO2, phthalic acid SiO2, phthalic acid, and SiO2 were then identified using infrared spectroscopy (IR). Particles were ground to flat wafers with KBr (FTIR grade) under pressure, and were characterized using IR spectroscopy (Perkin-Elmer Spectrum RX1 spectrometer, Canton, MA, USA).

Chemical Probe Conditions for MCF-7 and MDA-MB-231 Cell Lysates
The individual and triplicate MCF-7 and MDA-MB-231 cell lysates containing 100 μg of protein were incubated with APTES-modified (10 mg; probe 1) and phthalic acid-modified (10 mg; probe 2) chemical probes diluted to 400 μL in a phosphate-buffer saline (PBS) containing 2.7 mM KCl, 137 mM NaCl, 8 mM NaH2PH4, and 1.4 mM KH2PO4, at 37 °C for 4 h. After centrifugation, supernatants were removed and chemical probes were washed with 200 μL of PBS buffer and incubated at 37 °C for 4 h, three times.

Tryptic Digestion and Quantitative Dimethyl Labeling
After centrifugation, supernatants were removed and protein-bound chemical probes were eluted in 0.1% SDS. Before tryptic digestion, SDS was removed by TCA precipitation and the extracted proteins were reduced by DTT, alkylated by IAM, and digested using 0.2 μg trypsin. After 4 h, an additional 0.2 μg of trypsin was added and samples were incubated at 37 °C for a further 18 h. Bound

Establishment of STRING Protein-Protein Interaction Networks
Interactions between proteins and phthalic acid were predicted using the STRING database (version 9.1), and relationships between phthalate-related proteins and associated proteins were demonstrated.

Conclusions
In this study, phthalic acid chemical probes were used to demonstrate relationships between phthalic acid-sensitive proteins in MCF-7 and MDA-MB-231 cells. In these experiments, phthalate-like (phthalic acid) structures interacted with a group of proteasome subunit proteins in MCF-7 cells, and with a group of ribosomal proteins in MDA-MB-231 cells. Moreover, these proteins were connected by heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. Finally, protein networks in MCF-7 and MDA-MB-231 cells were established using STRING protein-protein interaction software. Future studies may identify phthalate receptors using chemical probes.