A Proteomic Strategy for Identification of Proteins Responding to

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A Proteomic Strategy for Identification of Proteins Responding to Cisplatin-Damaged DNA Wenjuan Zeng, Zhifeng Du, Qun Luo, Yao Zhao, Yuanyuan Wang, Kui Wu, Feifei Jia, Yanyan Zhang, and Fuyi Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00554 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Analytical Chemistry

A Proteomic Strategy for Identification of Proteins Responding to Cisplatin-Damaged DNA Wenjuan Zeng,ab Zhifeng Du,a Qun Luo,ab Yao Zhao,a Yuanyuan Wang,a Kui Wu,a Feifei Jia,a Yanyan Zhang*a and Fuyi Wang*abc Beijing National Laboratory for Molecular Sciences; National Centre for Mass Spectrometry in Beijing; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China. b University of Chinese Academy of Sciences, Beijing 100049, P. R. China. c Basic Medical College, Shandong University of Chinese Traditional Medicine, Jinan 250355, P. R. China a

ABSTRACT: A new proteomic strategy combining functionalized magnetic nanoparticle affinity probes with mass spectrometry was developed to capture and identify proteins specifically responding to 1,2-d(GpG) intrastrand cisplatin-crosslinked DNA, the major DNA lesion caused by cisplatin and thought to induce apoptosis. A 16-mer oligodeoxynucleotide (ODN) duplex and its cisplatin-crosslinked adduct were immobilized on magnetic nanoparticles via click reaction, respectively, to fabricate negative and positive affinity probes which were very stable in cellular protein extracts due to the excellent bio-orthogonality of click chemistry and the inertness of covalent triazole linker. Quantitative mass spectrometry results unambiguously revealed the predominant binding of HMGB1 and HMGB2, the well-established specific binders of 1,2-cisplatin-crosslinked DNA, to the cisplatincrosslinked ODN, thus validating the accuracy and reliability of our strategy. Furthermore, 5 RNA or single-stranded DNA binding proteins, namely hnRNP A/B, RRP44, RL30, RL13 and NCL, were demonstrated to recognize specifically the cisplatinated ODN, indicating the significantly unwound ODN duplex by cisplatin crosslinking. In contrast, the binding of a transcription factor TFIIFa to DNA was retarded due to cisplatin damage, implying that cisplatin lesion stalls DNA transcription. These findings promote understanding in the cellular responses to cisplatin-damaged DNA, and inspire further precise elucidation of action mechanism of cisplatin.

Cisplatin, a platinum-based anticancer agent, is one of the most common chemotherapy drugs for clinical treatment of solid tumors including testicular, ovarian, colorectal and nonsmall-cell lung cancer.1 As a DNA-damaging cytotoxic drug, cisplatin binds to DNA and forms interstrand and intrastrand (primarily 1,2-d(GpG) and 1,2-d(ApG) intrastrand) platinumDNA crosslinks, leading to partial unwinding and distortion in the DNA duplex conformation which blocks DNA replication and ultimately induces apoptosis.2-6 When DNA is damaged by cisplatin, damage-response proteins will recognize the lesions and subsequently activate corresponding signal pathways leading to cell cycle arrest. Then, DNA repair proteins will try to repair the lesions. If the repair fails, apoptosis will be initiated and the drug exhibits anticancer activity; if the repair succeeds, cells continue to survive and the drug loses effectiveness.7-9 Hence, fundamental understanding of cellular responses to cisplatin-damaged DNA could improve the effectiveness of the existing drugs and facilitate the design of novel metal-based anticancer agents with better performance. In order to explore the complicated cellular responses to DNA lesion by cisplatin, continuous efforts have been made to isolate and identify proteins that specifically respond to cisplatin-crosslinked DNA. In the early time, electrophoresis mobility shift assays (EMSAs) and southwestern blotting analysis were employed to identify proteins specifically binding to cisplatinated DNA.10-12 Hughes et al. developed an

affinity precipitation technique using cisplatin-crosslinked DNA cellulose and characterized a set of HeLaS3 nuclear proteins including high mobility group (HMG) proteins which specifically interact with cisplatin-crosslinked DNA.13 Later, a more systematic isolation of proteins recognizing DNA damage by cisplatin was performed by fractionating human cellular protein extracts through a sepharose column modified with cisplatin-damaged DNA.14 In 2004, on the basis of photoaffinity strategy using the cisplatin analogue containing a tethered, photoreactive benzophenone moiety that can crosslink the target proteins upon UV irradiation, poly[ADPribose] polymerase 1 (PARP-1) and high mobility group box (HMGB) proteins were identified to interact specifically with cisplatin-crosslinked DNA.15 Recently, affinity purification coupled with proteomics has been widely used to analyze protein complexes and study the protein-protein/DNA/RNA interactions in large scale.16 Some of the pull-down assays were performed using gel beads in combination with bovine serum albumin (BSA) to avoid nonspecific binding of high-abundance proteins.17 Consequently, gel electrophoresis is required to separate BSA from the captured target proteins.18 However, in-gel digestion reduced the sensitivity and throughput of these methods.19, 20 Streptavidin-coated magnetic beads were also extensively applied to isolated proteins due to virtue of the high affinity of biotin-streptavidin system.15, 21 Nonetheless, the interference of streptavidin also exists as there are numerous endogenous

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biotinylated proteins in cells which nonspecifically bind to the streptavidin on the beads, thereby interfering with the capture and detection of target proteins. Recently, our group developed a mass spectrometry (MS)-based proteomic approach coupled with functionalized gold nanoparticles (AuNPs) as affinity probes to capture and identify the proteins responding to platinated DNA.22 AuNPs have excellent watersolubility, large surface area and are easy to be functionalized. However, as AuNPs are generally modified with thiolcontaining molecules via the formation of Au-S bond to function as affinity probes, they might be unstable due to the possible competitive reaction of the cellular endogenous thiolcontaining species such as cysteine, glutathione and proteins. Additionally, during the functionalization of AuNPs with DNA fragments, high concentration of NaCl was required for the aging of AuNPs, which may cause dissociation of DNA bound platinum complexes.23, 24 To address this issue, in the present work we developed a new proteomic method in combination of magnetic nanoparticle affinity probe and quantitative mass spectrometry to capture and identify proteins responding to cisplatincrosslinked DNA. We immobilized the 1,2-d(GpG) intrastrand cisplatin-crosslinked oligodeoxynucleotides (ODNs) on the magnetic nanoparticles (MNPs) via click reaction to construct the affinity probes without needing high concentration of NaCl for aging, which were then incubated with the whole cell protein extracts in vitro to pull down proteins for subsequent tryptic digestion and MS analysis. With the virtues of the excellent bio-orthogonality of click chemistry and the generated inert covalent triazole linker, the affinity probes were very stable in the protein extracts. MS identification and quantification results showed that HMGB1 and HMGB2 were predominant among the captured proteins specifically responding to 1,2-crosslinked DNA, thereby validating the accuracy and reliability of this method. In addition, some other proteins such as heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B) and nucleolin (NCL) were also revealed to specifically respond to cisplatin-DNA crosslinks, while the binding of a transcription factor TFIIFa to DNA was significantly decreased due to cisplatin damage. These findings provide novel insights into better understanding in the cellular responses to cisplatin-damaged DNA.

EXPERIMENTAL SECTION Materials. Alkyne-modified Fe3O4@SiO2 core-shell magnetic nanoparticles (MNPs) was purchased from Enriching Biotechnology Ltd. (Shanghai, China). The 16-mer singlestranded oligodeoxynucleotide (ODN), 5CCTCTCTGGTCCTTCC-3 (I) and its azide-modified complimentary strand, N3-5-GGAGAGACCAGGAAGG-3 (N3-II), were obtained from TaKaRa (Dalian, China). Tris[(1benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), Phenylmethanesulfonyl fluoride (PMSF) and N-acetoxy-D3succinimide (D3-NAS) were purchased from Sigma-Aldrich (USA). N-acetoxy-H3-succinimide (H3-NAS) was obtained from J&K (Shanghai, China). All primary and secondary antibodies, rabbit monoclonal anti-human HMGB1 (ab79823), rabbit monoclonal anti-human HMGB2 (ab124670), rabbit polyclonal anti-human β-actin (ab8227) and horseradish peroxidase-conjugated secondary antibody (ab6721), were purchased from Abcam (Cambridge, MA, USA). De-ionized water from Millipore system was used throughout the experiments.

Preparation of platinated oligonucleotides. Cisplatin (Figure 1a, Beijing Ouhe technology, China) was incubated with the 16-mer single-stranded ODN I with a molar ratio of 0.8: 1 at 310 K for 3 days. Then, the platinated oligonucleotide was purified by high-performance liquid chromatography (HPLC) using a C18 column (4.6  150 mm, 5 m, Shiseido) on an Agilent 1200 HPLC system. The mobile phase consisted of water with 20 mM TEAA (solvent A) and acetonitrile with 20 mM TEAA (solvent B). The isolated cisplatin crosslinked strand I was characterized by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) (Autoflex III, Bruker) in positive mode (Figure S1). After lyophilized, the cisplatin-crosslinked ODN I was redissolved in water and quantified by UV-visible spectrometer, and then annealed with equal amount of the 5azide-modified complementary strand II (N3-II) in the binding buffer (10 mM Tris, 50 mM NaCl, pH 7.50) to afford the cisplatin-crosslinked duplex III (N3-cisPt-III).

Figure 1. (a) The chemical structure of cisplatin. (b) The sequence of the intact ODN duplex (N3-III) with -GpG- motif on the top strand, and the 1,2-d(GpG) intrastrand cisplatincrosslinked ODN duplex (N3-cisPt-III) with the -GG- motif crosslinked by cisplatin. The bold magenta letters indicated the platinated bases. (c, d) Schematic diagrams of the MNP-III negative probe (c) and MNP-cisPt-III positive probe (d).

Preparation and characterization of MNP-ODN probes. Alkyne-modified Fe3O4@SiO2 core-shell MNPs (33 L, 30 mg/mL) were washed five times with water and mixed with 1.0 nmol intact ODN duplex (N3-III) or 1,2-d(GpG) intrastrand cisplatin-crosslinked ODN duplex (N3-cisPt-III) (Fig. 1b) in 320 L binding buffer (10 mM Tris, 50 mM NaCl, pH 7.50). Then, 4 L of TBTA (10 mM), sodium ascorbate (50 mM) and CuSO4 (5 mM) were added into the mixture, respectively. After homogenously mixing, the reaction mixture was shaken at room temperature for 1 h. The fabricated probes were separated with a magnet and washed with 350 L phosphate buffered saline with Tween 20 (PBST, 0.02%) for 20 min. Transmission electron microscopy (TEM), UV-visible spectroscopy, and time of flight secondary ion mass spectrometry (ToF-SIMS) were used to characterize the morphology of the probes, the number of bound N3-III or N3cisPt-III molecules on the probes and the surface chemical compositions of the probes, respectively. Cell culturing. Human breast cancer cells MCF-7 (National Infrastructure of Cell Line Resource, Beijing, China) were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). Cells were grown at 5% CO2 in a humidified incubator at 37 ˚C.


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Analytical Chemistry Protein extraction. MCF-7 cells were harvested, washed with PBS and collected by trypsin (Gibco), followed by washing and centrifuging with PBS for three times. Cells were lysed in 250 L protein extraction solution (Bioteke, China) with 1 mM PMSF (Sigma) and 1 L protease inhibitor cocktail (BestBio Science, China) for 2 h on ice. The obtained cell lysate was centrifuged at 14000 g at 4 ˚C for 8 min and the supernatant containing proteins was transferred to a clean tube. The concentration of extracted proteins was determined by bicinchoninic acid (BCA) assay kit (Tiangen, China). Protein affinity pull-down. The MNPs modified with N3III or N3-cisPt-III were incubated with 100 g whole cell protein extracts to pull down proteins on ice for 4 h. The MNPs with captured proteins were separated by a magnet and washed with PBS for five times. Sample preparation for MS analysis. The captured proteins were eluted from MNPs by 6 M urea, reduced with 10 mM dithiothreitol (DTT) at 37 ˚C for 2 h and then alkylated with 30 mM iodoacetamide (IAA) at room temperature in the dark for 1 h. The protein solution was transferred to a Microcon YM-3 filter (Millipore) and ultrafiltered three times for 20 min each by adding 20 mM NH4HCO3 (pH 8.50) for desalting and buffer exchange. After ultrafiltration, the proteins were digested with 1 g trypsin at 37 ˚C for 16 h. The stable isotope labeling reactions were carried out following the procedure reported previously.22, 25-27 Briefly, a 100-fold molar excess of H3-NAS and D3-NAS were added to the digests of proteins captured by MNP-III (negative) and MNP-cisPt-III (positive) probes, respectively, and the reactions proceeded at room temperature for 5 h. After completion of the labeling reactions, equal aliquots of negative and positive samples were combined and treated with N-hydroxylamine at room temperature and pH 10 – 11 for 20 min to eliminate the excess of H3-NAS and D3-NAS. The labeled peptide mixture was lyophilized, re-dissolved in 30 L water containing 0.1% formic acid and then desalted with Ziptip C18 (Millipore). The desalted peptide mixture was dried under vacuum and stored at -80 ˚C before mass spectrometry analysis. MS measurements. The desalted isotopically labeled peptide mixture obtained as described above was re-dissolved in 5 L water with 0.1% formic acid and injected into a Dionex Ultimate 3000 RSLCnano system (Dionex, Germany). The peptides were concentrated by a C18 trap column (75 m  2 cm, 3 m, ThermoFisher Scientific) and then separated by a C18 analytical column (75 m  15 cm, 2 m, ThermoFisher Scientific). The mobile phase consisted of water/acetonitrile (95/5, v/v) with 0.1% formic acid (solvent A) and acetonitrile/water (95/5, v/v) with 0.1% formic acid (solvent B). An isocratic flow at 5 L/min for 10 min with solvent A alone was used for the peptide concentration by the C18 trap column and then a linear gradient from 1% to 45% solvent B over 150 min at a flow rate of 300 nL/min was used for the peptide separation by the C18 analytical column. The RSLCnano system was coupled to a Xevo G2 QTOF electrospray ionization mass spectrometer (ESI-MS, Waters) equipped with a nanospray source. The data acquisition was performed in the data-dependent mode with a survey scan (m/z 350 – 1700) followed by MS/MS scans of the top 8 intensive precursor ions with charge states of 2+, 3+ or 4+. Precursor ions were filtered using a 20 s dynamic exclusion window.

MS data analysis. Mascot distiller 2.5 with the in-house MASCOT 2.5.1 search engine was used for peak detection from the raw MS data, and protein identification and quantification. The search was performed on Swiss-Prot 2018.04 (human) protein database (557275 sequences; 20341 human protein sequence entries). The search parameters included up to 1 missed cleavage for tryptic digestion, a precursor ion mass tolerance of 30 ppm, a product ion mass tolerance of 0.5 Da, carbamidomethylation (cysteine) as a fixed modification, oxidation (methionine) and acetylation (protein N terminus) as variable modifications. The quantification method based on the relative intensities of extracted ion chromatograms (XICs) for precursor ions with acetylation or deuterioacetylation of N terminus and lysine was also added to the search parameters. The quantification ratios of proteins were given by the weighted average of the intensity ratios of peptide ions from the same protein in positive and negative samples. Bioinformatics analysis. The venn diagram and common proteins of the three biological replicates were obtained using the on-line tool VENNY 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/). Heat maps of MS quantitation ratios of the common proteins from three biological replicates were generated by HemI (version 1.0). The common proteins were uploaded to the Search Tool for the Retrieval of Interacting Genes (STRING, version 10.5, https://string-db.org/) database to generate the protein-protein interaction (PPI) network and the combined score ≥ 0.7 was set as the criterion to obtain the interaction information with high confidence. Then, the PPI information was imported into Cytoscape (version 3.6.1) to construct and modify the PPI network. Gene Ontology (GO) information, including biological process, molecular function, cellular component of the proteins with large MS quantitation fold change, was exported from STRING database and refined with reference to the Uniprot database. Western blotting. The same amount of the whole cell protein extracts and the captured proteins by negative and positive probes were individually boiled with the gel-loading buffer, and loaded onto a 4 – 12% gradient SDS-PAGE gel (Genscript, Nanjing, China) for electrophoresis. The separated proteins were transferred to PVDF membrane (Millipore, 0.2 m). The membrane was blocked in 5% nonfat dry milk in 0.1% PBST at room temperature for 1 h and then incubated with primary antibodies in appropriate dilutions at 4 ˚C overnight. The blots were washed four times with PBST for 5 min each and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After washed with PBST, the protein bands were visualized by enhanced chemiluminescence (Beyotime, China) and the optical densities were quantified using an image analyzer (Tanon 5200Multi, China).

RESULTS The construction and characterization of magnetic nanoparticle affinity probes. The 1,2-d(GpG) intrastrand cisplatin-crosslinked oligodeoxynucleotide duplex III bearing an azide tag (N3-cisPt-III, Fig. 1a-1b) was prepared and characterized by MALDI-TOF-MS (Figure S1 in the Supporting Information). Then, the intact duplex III tagged an azide group (N3-III) and the N3-cisPt-III complex were covalently immobilized on the alkyne-modified Fe3O4@SiO2



core-shell MNPs via click reaction to construct the MNP-III (Figure 1c) and MNP-cisPt-III (Figure 1d) affinity probes, respectively. The UV absorption spectra (Figure S2a) showed that the click reaction was completed within 1 h and the loaded duplex III on the MNPs was determined to be 1.26 nmol/mg. As shown in the TEM image in Figure S2b, the diameter of a bare MNP was approximately 150 nm. After the conjugation of duplex III, no significant increase was observed for the diameter of MNPs due to the short length of ODNs compared with MNPs, but the peripheral SiO2 shell became thicker and smoother (Figure S2c). Moreover, time of flight secondary ion mass spectrometry (ToF-SIMS) was employed to examine the surface chemical compositions of bare MNPs and MNP-cisPt-III. Mass spectra (Figure S3) of fragment ions C2N3- arising from triazole moiety formed via click reaction, C5H4N5- from adenine in ODNs and PtCN- from cisplatin-ODN adduct further confirmed that cisPt-III was indeed immobilized on MNPs via click reaction. Taken together, UV-visible absorption spectroscopy, TEM and ToFSIMS analysis demonstrated the successful construction of the magnetic nanoparticle affinity probes through click reaction. Pull-down and identification of proteins recognizing the 1,2-d(GpG) intrastrand cisplatin-crosslinked ODN. The cisplatin crosslinked ODN modified MNPs (MNP-cisPt-III) functioned as the positive probes (designated as Pos) to capture the proteins specifically recognizing the cisplatincrosslinked DNA. Meanwhile, MNPs modified by the intact duplex III (MNP-III) were used as negative probes (designated as Neg) to discriminate the proteins responding to cisplatin-DNA adducts from general DNA-binding proteins. Both the negative and positive probes were incubated with 100 g protein extract of human breast MCF-7 cancer cells to perform the pull-down experiments, respectively.

Figure 2. Schematic illustration of protein capture, identification and quantification by the proteomic method based on magnetic nanoparticle affinity probes and mass spectrometry.

Then, we employed a quantitative proteomic method based on mass spectrometry and stable isotope labeling with acetylation reagents to identify the proteins specifically recognizing the cisplatin crosslinked ODN. The proteins

pulled down by Neg and Pos probes were eluted and digested by trypsin, separately. The obtained peptides derived from the pulldowns by Neg (i.e. negative sample) were then “lightlabeled” by N-acetoxy-H3-succinimide (H3-NAS) and those by Pos (i.e. positive sample) were “heavy-labeled” by N-acetoxyD3-succinimide (D3-NAS). The labeled negative and positive peptides were 1: 1 mixed and analyzed by tandem mass spectrometry coupled with nano-LC (nanoLC-MS/MS). The whole experimental workflow for protein capture, identification and quantification was displayed in Figure 2. The pull-down experiments were carried out in three replicates independently, and 168 proteins in common were identified (Figure 3a). The detailed MS data are provided in the Supporting Information as Table S1. The MS quantitation results were shown as heavy-to-light (HPos/LNeg) ratios. The similar ratios of the same protein in the three replicates (Figure 3b) indicate good reproducibility and reliability of the results. The HPos/LNeg ratios of the 168 identified proteins distributed in a wide range of 0.00 – 11.00 (Figure 3c), but most of which (136 proteins) were concentrated between 1.50 and 0.67 (reciprocal of 1.50), showing no significant difference in their binding affinity between the intact and cisplatin-crosslinked ODN III. Seven proteins with HPos/LNeg ratios ≥ 2.00 were demonstrated to respond specifically to the cisplatin damaged ODN (Figure 3b). Among them, 5 proteins were differentially enriched by Pos with HPos/LNeg ratios over the range of 2.00 – 3.00, including hnRNP A/B, RRP44 (exosome complex exonuclease RRP44), RL30 (60S ribosomal protein L30), RL13 and NCL (Figure 3b). Particularly, HMGB1 and HMGB2 were found to have high HPos/LNeg ratios (10.48 and 9.05, respectively), indicating the predominant binding affinity of these two HMG proteins to the cisplatin damaged ODN. The representative MS/MS spectra of tryptic peptides from HMGB1 and HMGB2 captured by Pos are displayed in Figure 4a-b, and the quantitative MS spectra for the corresponding peptides deriving from HMGB1 and HMGB2 captured by the negative and positive probes are shown in Figure 4c-d, respectively. The captured HMGB1 and HMGB2 by positive and negative probes were further analyzed by Western Blotting (WB, insets in Figure 4c-d). The WB optical density ratios of HMGB1 and HMGB2 pulled down by Pos and Neg were 8.62 and 8.05, respectively, being consistent to those determined by MS quantification. Apart from the proteins enriched by the positive probe, surprisingly, TFIIFa (TFIIFα or RAP74), with a HPos/LNeg ratio of 0.34 (≤ 0.50, reciprocal of 2.00) was enriched by the negative probe, i.e. the intact ODN III (Figure 3b). This indicates a decreased affinity of TFIIFa to DNA due to the alteration of DNA conformation caused by cisplatin damage. Bioinformatics analysis. In order to deeply study the biological functions and interactions of the identified proteins, the protein-protein interaction (PPI) network of the 168 common proteins captured by the intact and cisplatin crosslinked ODN duplex III was generated by STRING 10.5, visualized and modified in Cytoscape 3.6.1 (Figure S4). The PPI network was complicated and consists of 152 nodes (i.e. proteins) connected by 1390 edges (i.e. interactions), which suggests close interactions among the captured proteins. These proteins are classified into four clusters according to their


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Figure 3. (a) Venn diagram of captured and identified proteins from three biological replicates by negative (Neg) and positive (Pos) probes. (b) Heat map generated from HPos/LNeg ratios of the 168 common proteins determined by MS on three biological replicates. Lane 1, 2 and 3 corresponded to each replicate. (c) Histogram of the distribution of HPos/LNeg ratios of the identified 168 common proteins.

biological functions and interactions: cytoskeletal proteins, heterogeneous nuclear ribonucleoproteins, and proteins involved in protein translation and DNA damage response. Close connections within and between the four clusters are apparently displayed. In the DNA damage response proteins cluster, HMGB1 and HMGB2 were prominent and noticeable due to their large HPos/LNeg ratios (Figure 3b). Besides, HMGB1 was shown to be one of the first neighbors of TFІIFa in the PPI network, suggesting that there were strong and direct interactions between the two proteins (Figure S5). Moreover, the gene Ontology (GO) information of proteins with HPos/LNeg ratios larger than 1.50 or smaller than 0.67 were listed in Table S2 in the Supporting Information. The GO information suggests that except for HMGB1 and HMGB2, which are well-known proteins responding to DNA damage by cisplatin, the majority of the proteins which showed higher affinity to the cisplatin crosslinked duplex III than to the intact duplex III are RNA binding, or single-stranded DNA (ssDNA) binding proteins. This implies that the intrastrand crosslinking by cisplatin causes unwinding of the DNA target, making the double-stranded DNA behave as RNA or ssDNA.

DISCUSSION Accuracy and reliability of proposed proteomic strategy. In this work, we have developed a new proteomic method by combining magnetic nanoparticle affinity probes and quantitative mass spectrometry to capture and identify proteins responding to DNA lesion by cisplatin. The successful capture and identification of HMGB1 and HMGB2 was well consistent with previous reports that the high mobility group proteins could specifically recognize the 1,2-intrastrand cisplatin-DNA crosslinks,15, 28-30 validating the accuracy and reliability of our proteomic method. Due to the excellent bio-orthogonality of click reaction through azide-alkyne and the generated inert covalent triazole linker, the affinity probes were very stable to withstand the

interference arising from other species in the protein extracts, including thiol-containing amino acids, peptides and proteins. Moreover, the probes were easy to be separated mildly from the solution by a magnet in a few seconds without the need of high-speed centrifugation, enabling the preservation of the weak interactions between protein-protein and protein-DNA complexes so as to fully pull down targeted protein-protein complexes from protein extracts. Using functionalized gold nanoparticle probes to capture proteins responding to 1,2d(GpG) crosslinked ODN by cisplatin, we have previously identified only 33 proteins interacting with cisplatin crosslinked ODN, of which HMGB1 was the only one which specifically bound to cisplatin crosslinked ODN with 5.90-fold higher affinity in comparison with binding to the same intact ODN.22 Using the new proteomic strategy, we identified 168 proteins binding to the cisplatin-damaged ODN, of which 7 proteins, including HMGB1 and HMGB2, showed significant higher affinity (>2-fold) to the cisplatinated ODN than to the intact ODN. Moreover, we have herein identified for the first time other five proteins which are RNA binding and/or singlestranded DNA binding proteins, and showed specific interaction with the cisplatinated ODN.1, 29-32 It is notable that a transcription factor, i.e. TFIIFa, was revealed also for the first time to have lower affinity (< 0.50) to the cisplatinated ODN than to the intact one.1, 29-32 These results further demonstrated the high efficiency of our new proteomic strategy for capturing and identifying proteins which respond to DNA damage by cisplatin. Biological insights into the cellular responses to cisplatin-damaged DNA. HMGB1 and HMGB2 are two members of high mobility group proteins, which are highly conserved non-histone chromosomal proteins preferentially binding to DNA with bent or distorted conformation.1, 33 They participate in the regulation of numerous nuclear processes including transcription, recombination, replication and chromatin remodeling.1 HMGB1 and HMGB2 are well-known



to specifically recognize cisplatin-crosslinked DNA, and have been reported to be involved in the cisplatin mechanism of action.1, 34, 35 In particular, in vitro studies demonstrate that HMGB1 can inhibit nucleotide excision repair (NER) of cisplatinated DNA presumably by binding to the lesion sites and then shielding it from recognition by the DNA damage sensors.36, 37 Moreover, a number of studies have correlated the expression level of HMGB1 and HMGB2 to cisplatin sensitivity and resistance.38-40 The predominant binding of HMGB1 and HMGB2 to cisplatin-crosslinked ODN duplex III in this work further verify that HMGB proteins plays an important role in conveying the antitumor activity of cisplatin.

Figure 4. Representative MS/MS spectra of a tryptic peptide of HMGB1 (a) and HMGB2 (b) captured by positive probe. Quantitative MS spectra for a peptide of HMGB1 (c) and HMGB2 (d) captured by negative probe (black) and positive probe (red). Western blotting images of HMGB1 (inset in (c)) and HMGB2 (inset in (d)) in protein extracts of MCF-7 cells, and those pulled down by positive (Pos) and negative (Neg) probes from the protein extracts, respectively. β-actin was used as the internal reference.

Apart from HMGB1 and HMGB2, 5 other proteins, hnRNP A/B, RRP44, RL30, RL13 and NCL, were identified to be significantly enriched by the positive probe, i.e. the cisplatin crosslinked ODN III, which all possess RNA-binding function (more details are given in the Supporting Information). These proteins have not been previously reported to interact specifically with cisplatin-DNA intrastrand crosslinks, and the discoveries herein imply that they may also play important roles in cellular response to DNA damage by cisplatin. Recently, an increasing number of RNA binding proteins have been demonstrated to be involved in DNA damage response, designated as DNA-damage response RNA-binding proteins (DDRBPs).41-43 DNA lesions attract DDRBPs which may be post-translationally modified by enzymes at the damage sites

or directly participate in the damage response, signaling, repair and chromatin modifications.43, 44 Therefore, DDRBPs act as both regulators and regulated factors of DNA damage response. Besides the 5 proteins described above with HPos/LNeg ratios greater than 2.00, we found that 22 proteins with HPos/LNeg ratios between 1.50 and 2.00 also showed slight enrichment by the positive probe. These proteins, for example, DDX1 (ATPdependent RNA helicase DDX1), GRWD1 (Glutamate-rich WD repeat-containing protein 1), HNRDL (Heterogeneous nuclear ribonucleoprotein D-like,) and PC4 (Activated RNA polymerase II transcriptional coactivator p15), are involved in either transcription regulation or DNA damage repair (more detailed discussions are shown in the Supporting Information).22, 45-48 However, further investigation and evidence are needed to elucidate the precise role of the above proteins in cisplatin-DNA damage response. In contrast with the enriched proteins by the positive probe mentioned above, we demonstrate herein that TFIIFa showed significantly reduced affinity to the cisplatin-crosslinked ODN compared with the intact one. TFІІFa is the large subunit of transcription factor II F (TFIIF), and has been shown to participate in both the initiation and elongation stages of transcription.49, 50 TFІІFa binds directly to RNA polymerase II (pol II) and several transcription factors (TFІІFb, TFIIB, TFIIE56, etc.), and may coordinate the assembly of the preinitiation complex like TBP-TFIIB-TFIIF-pol II-TFIIE complex through the substantial protein-protein interactions and recruitment of other transcription factors.51, 52 Therefore, TFІІFa is an important member of transcription machinery. Given that cisplatin-DNA crosslinks inhibit transcription,1 we hypothesized the binding affinity of TFІІFa or even the whole transcription machinery to DNA was decreased possibly due to the cisplatin obstacle or the alteration of DNA conformation caused by cisplatin damage. Moreover, there are significant interaction between TFІІFa and HMGB1. Thus, it is possible that HMGB1 competes with TFІІFa for DNA binding or removes it from cisplatin-damaged DNA as HMGB1 has high affinity to DNA with bent or distorted structures. In addition to TFІІFa, other 2 proteins, C2AIL (CDKN2Ainteracting protein N-terminal-like protein) with MS ratio 0.62 and RS20 (40S ribosomal protein S20) with MS ratio 0.67 showed slightly reduced affinity to the cisplatin-crosslinked ODN. C2AIL is located in nucleolus and contains a putative XRN2-binding domain (XTBD) which interacts with XRN2, an exoribonuclease, functioning in transcription termination and RNA processing and degradation.53 RS20 is a ribosomal protein which binds to RNA and participates in translation initiation. The roles of C2AIL and RS20 in response to cisplatin damage remain unclear, and further investigations are needed to clarify the underlying mechanisms. Different from previous reports which mainly focused on proteins specifically recognizing the cisplatin-DNA adducts,1, 29-32 our observations of the proteins showing lower affinity to DNA alerts us of the existence and importance of them in response to cisplatin damage.

CONCLUSION Cellular responses to cisplatin-damaged DNA are closely associated with cisplatin efficiency and resistance. A better understanding in the cellular responses to cisplatin-damaged DNA would aid in precise elucidation of the mechanism of


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Analytical Chemistry action of cisplatin and improve the drug effectiveness. In this work, we have developed a new and efficient proteomic method in combination of magnetic nanoparticle affinity probe and mass spectrometry to effectively capture and identify proteins responding to 1,2-d(GpG) intrastrand cisplatincrosslinked DNA. Among the identified proteins, HMGB1 and HMGB2 were predominant with the largest MS quantitation ratios, which were in good consistency with the previously well-established results and thus validated the accuracy and reliability of our method. More importantly, the application of our proposed method discovered some RNA and/or singlestranded DNA binding proteins such as hnRNP A/B, RRP44, RL30, RL13 and NCL specifically responding to cisplatinDNA intrastrand crosslinks, implying that cisplatin damage on double-stranded DNA unwinds DNA duplex, and makes more RNA- and single-stranded DNA binding proteins interact with cisplatin lesion sites. Moreover, we also found that the binding of transcription factors TFIIFa to DNA was significantly reduced due to cisplatin damage. These unique findings inspire further studies on the effect of the altered binding affinity of these proteins to DNA due to cisplatin damage on effectiveness of cisplatin. Our studies also demonstrate that DNA-conjugated magnetic nanoparticles constructed via click chemistry have great potential to capture proteins responding to damaged DNA in vitro. Our new strategy has a number of notable advantages: (1) MNPs allow fast and mild separation by a magnet in a few seconds without high-speed centrifugation which well preserves weak interactions between proteinprotein and protein-DNA complexes and thereby enables the efficient pull-down of intact large protein complexes from cellular protein extracts; (2) the affinity probes are highly stable to withstand the interference of other species in the protein extracts thanks to the excellent bio-orthogonality of click chemistry and the generated inert covalent triazole linker; and (3) intact oligodeoxynucleotide duplex is used as negative control to exclude general DNA-binding proteins which does not specifically recognize cisplatin-damaged DNA. Collectively, this proteomic strategy is versatile and efficient. We anticipate its broad application in the identification of proteins in other protein-protein and/or protein-DNA/RNA interaction networks. For example, cisplatin was also reported to bind with RNA and forms Pt-RNA adducts, which might interfere with RNA-based processes. However, the detailed cellular responses to Pt-RNA adducts remain largely unknown,54, 55 and could be investigated using our proteomic strategy. Moreover, in our lab we are applying this method to investigate the molecular mechanisms underlying the acquired resistance of cancer cells to cisplatin.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterization results of the MNP probes, PPI networks, detailed MS quantitative data and GO information of identified proteins specifically responding to DNA damaged by cisplatin are provided as Figures S1-S5 and Table S1-S2 (PDF)

AUTHOR INFORMATION Corresponding Author

* E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the NSFC (Grant Nos. 21575145, 21635008, 21621062, 21790390 and 21790392) for support.

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* E-mail: [email protected]



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A Proteomic Strategy for Identification of Proteins Responding to

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