Graphene Oxide Facilitated Comprehensive Analysis of Cellular

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Biological and Medical Applications of Materials and Interfaces

Graphene Oxide Facilitated Comprehensive Analysis of Cellular Nucleic Acid Binding Proteins for Lung Cancer Zhi Shang, Liqiang Qian, Sha Liu, Xiaomin Niu, Zhi Qiao, Yan Sun, Yan Zhang, Liu-Yin Fan, Xin Guan, Cheng-Xi Cao, and Hua Xiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05428 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Applied Materials & Interfaces

Graphene Oxide Facilitated Comprehensive Analysis of Cellular Nucleic Acid Binding Proteins for Lung Cancer

Zhi Shang1,†, Liqiang Qian2,†, Sha Liu1, Xiaomin Niu2, Zhi Qiao1, Yan Sun1, Yan Zhang3, Liu-Yin Fan1, Xin Guan4,*, Cheng-Xi Cao1,5,*, Hua Xiao1,*

1

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,

Shanghai Jiao Tong University, Shanghai, 200240, China. 2

Department of Shanghai Lung Cancer Center, Shanghai Chest Hospital, Shanghai Jiao Tong

University, Shanghai, 200030, China. 3

School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.

4

Department of Thoracic Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of

Medicine, Shanghai, 200011, China. 5

Department of Instrument Science and Engineering, School of Electronic Information and Electrical

Engineering, Shanghai Jiao Tong University, Shanghai 200240, China

1

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ABSTRACT Nucleic acid binding proteins (NABPs) mediate a broad range of essential cellular functions. However, it is very challenging to comprehensively extract whole cellular NABPs due to the lack of approaches with high efficiency. To this end, carbon nanomaterials, including Graphene Oxide (GO), Carboxylated Graphene (cG), and Carboxylated Carbon Nanotube (cCNT), were utilized to extract cellular NABPs in this study through a new strategy. Our data demonstrated that GO, cG, and cCNT could extract nearly 100% cellular DNA in vitro. Conversely, their RNA extraction efficiency was 60%, 50%, and 29%, respectively, partially explain why GO has the highest NABPs yield than cG and cCNT. We further found that ionic bond mediated by cations between RNA and functional groups of nanomaterials facilitated RNA absorption on nanomaterials. About 2400 proteins were successfully identified from GO enriched NABPs sample and 88% of annotated NABPs were enriched at least 2 times when compared with cell lysate, indicating the high selectivity of our strategy. The developed method was further applied to compare the NABPs in two lung cancer cell lines with different tumor progression ability. According to label-free quantification results, 118 differentially expressed NABPs were discovered, and 6 candidate NABPs including ACAA2, GTF2I, VIM, SAMHD1, LYAR, and IGF2BP1 were successfully validated by immunoassay. The level of SAMHD1 in the serum of lung cancer patients was measured, which significantly increased upon cancer progression. Our results collectively demonstrated that GO is an ideal nanomaterial for NABPs selective extraction, which could be broadly used in varied physiological and pathophysiological settings. 2


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Keywords: Nucleic acid binding proteins / Carbon nanomaterials / Graphene Oxide / Selective extraction / Proteomics

INTRODUCTION Nucleic acid binding proteins (NABPs), namely DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), regulate many cellular processes, including DNA replication, transcription, translation, gene silencing, and RNA processing1-5. Defects in NABPs expression and function will lead to numerous diseases, including neuropathies, metabolic disorders, and cancer 6-7. Owing to their preeminent biological functions, the NABPs proteome as well as their activity states are highly informative for the cellular system. In the past decade, biochemical approaches have been used to characterize proteins binding on nucleic acids, including chromatin immunoprecipitation (ChIP) 8, electrophoretic mobility-shift assay (EMSA)

9

, DNA or RNA affinity extraction

10-11

. These approaches are either

gene-centered, in which an individual protein is used to identify target sequences, or protein-centered, in which a DNA sequence is used to screen for uncharacterized DBPs. Recently, several large-scale gene-centered approaches have been employed to profiling DBPs or RBPs. For instance, protein-binding microarray combined with bioinformatics has been used for protein-DNA interactions profiling in vitro, which identified 17,718 protein-DNA interactions between 460 DNA motifs predicted to bind DBPs and 4,191 human proteins of various function classes

12

. In addition, DNA concatenated tandem array was applied for cellular DBPs profiling

through incubating nuclear extract with a synthetic DNA containing a hundred consensus 3



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transcription factor binding elements, by which 878 DBPs and 941 DBPs were identified from cell lines and mouse tissues, respectively

13-14

. However, they cannot represent the global

information of cellular DBPs and might lead to some artificial interactions. With respect to RBPs extraction, mRNA immunoprecipitation has been used for high throughput RBPs purification. RBPs were covalently bounded to mRNA by using in vivo UV cross-linking, and captured with oligo (dT) beads. After RNase digestion and mass spectrometry analysis, 860 RBPs were identified

15

. Unfortunately, the mRNA isolation efficiency is low and will lose the information

of proteins, which bind to rRNA and tRNA. Additionally, these approaches are either laborious or complicated in general. Until now, no general methods were developed for large scale NABPs extraction, and it is still very challenging to develop an efficient while a facile method for complete extraction and identiﬁcation of mammalian NABPs. Usually, most NABPs are in low abundance in cells, enrichment of NABPs selectively and effectively will benefit downstream proteomics and molecular biology research. Since NABPs are binding on nucleic acids, it is therefore feasible to extract NABPs through isolating nucleic acids. The traditional method for total nucleic acids isolation is based on organic solvent, which would lose most of the NABPs during the extraction procedure

16

. Due to the lack of methods

with high efficiency to isolate integrated nucleic acid associated proteins from cells, the global analysis of cellular NABPs is still a big challenge. Nanomaterials, such as Graphene (G), Graphene Oxide (GO), Carbon Nanotube (CNT), consist of sp2-conjugated atomic carbon, have stimulated intense research interests in biomedical 4


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research because of their outstanding mechanical

17

, thermal

18

, and electrical properties

19

.

Previously, G, GO, CNT were reported to adsorb single-stranded DNA (ssDNA) stably through the strong π-π stacking interaction of nucleotides with nanomaterials

20-21

, and absorbed ssDNA

can be detached by adding complementary ssDNA strands, because of the weak binding affinity between double-stranded DNA (dsDNA) and nanomaterials

21-22

. While most of the ssDNA and

dsDNA were synthesized by short DNA oligomer (30-90 bases), few studies focused on cellular intact DNA or RNA extraction, and the mechanism of nucleic acids absorption by nanomaterials is still not fully understood. Zhang et al. had extracted cellular chromatin using magnetic oxidized CNTs with nearly 95% DNA extraction efficiency and obtained 3790 proteins (2595 protein groups) with in vitro extraction 23. In addition, 4118 proteins were identified from living cells through their CNT-based in vivo extraction strategy

24

. CNT-based NABPs extraction is

very simple to perform. However, the selectivity of NABPs extracted is low, only 12% (459) and 15% (620) identified proteins have the annotation of NABPs for in vitro and in vivo extraction strategy, respectively. It has been demonstrated that the morphology of nanomaterials would significantly affect their biological performances. Two-dimensional (2D) nanosheets, including G and GO, show many unique properties because of their ultrathin thickness and ultrahigh carrier mobility than one-dimensional nanotubes, which enable them as very promising nano-platforms for bioapplications

25-26

. GO, the oxidized counterpart of G, contains functional groups such as

epoxide, carboxyl, and hydroxyl groups, which can undergo covalent, electrostatic, or hydrogen

5



bonding with proteins and other biomolecules. GO has shown great advantages in drug delivery 27

, gene transportation 28, biosensing 29, when compared to other conventional nanomaterials. In

the field of nucleic acid binding, most studies focus on the interactions between GO, G, CNT, and ssDNA individually, while there is no study related to the comparison of different nanomaterials with functional groups or dimensionality in nucleic acids as well as NABPs extraction. There is a critical need to further reveal the interaction mechanism of nanomaterials with nucleic acids. In the present study, we compared the ability of cG, GO, cCNT in cellular nucleic acids extraction, including DNA and RNA extraction, as well as corresponding NABPs enrichment. We developed a new strategy for NABPs isolation that has higher selectivity than the one used in the reference

23

. The mechanism behind the high efficient extraction of NABPs was also

discovered. The optimized method was further applied to enrich the NABPs in different lung cancer cell lines. The NABPs profiling in these cell lines were comprehensively compared through employing label-free quantification approach. Cancer related NABPs were discovered and verified through immunoassay. Their potential role in cancer research was further evaluated and discussed.

EXPERIMENTAL METHODS Carbon Nanomaterials and reagents

6


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G dispersion with diameter less than 500 nm, cG dispersion with diameter less than 500 nm, GO dispersion with diameter less than 500 nm, cCNT dispersion with diameter 20-30 nm and 0.73% carboxyl content, and hcCNT dispersion with diameter 10-20 nm and 2% carboxyl content were purchased from XF NANO, INC (Nanjing, China, http://en.xfnano.com/), which have been comprehensively

characterized

(Representative

characterization

data

are

shown

in

Supplementary Information for peer review). Benzonase nuclease, SDS, Urea, EDTA, EGTA, PMSF, Dithiothreitol (DTT), Iodoacetamide (IAA), PBS, Guanidine Hydrochloride, NH4HCO3, Acetone, and Acetic acid were purchased from Sigma (St. Louis, MO, USA). DAPI and cocktail of proteinase inhibitors were purchased from Roche (Mannheim, Germany). Trypsin (sequencing grade-modified) was purchased from Promega (Madison, WI, USA) and standard protein ladder was from Life Technologies (Carlsbad, CA, USA). DNase I and RNase A were obtained from Thermo Scientific (Rochford, IL, USA). Cell culture, cell nuclei isolation, and cell lysis The 293T cells, BEAS-2B cells, H460 cells, and H1299 cells were obtained from American Type Culture Collection (Manassas, VA, USA), which were grown in DMEM medium with 10% bovine serum (Gibco, USA) and 5% CO2 atmosphere at 37 ℃. Cells were split as they reached 90% confluency. The nuclear and cytoplasmic protein extraction kit (Transgen Biotech, Beijing, China) was employed to isolate the nuclei of 293T cells. To prepare total cell lysate for western blotting, 200 µL RIPA lysis buffer (150 mM NaCl, 1.0% Triton x-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) was used to lyse ca. 5×106 cells, then cell 7



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lysates were treated with sonication (BILON-1000Y, Shanghai, China) with 5 strikes (working for 2 seconds and pausing for 2 seconds) at 400 W. Protein concentrations were measured using BCA Protein Assay Kit (Thermo Scientific, Rochford, IL, USA). Serum sample collection Serum samples were collected according to approved protocols (IRB#M15017) by Institutional Review Board (IRB) of Shanghai Jiao Tong University from Shanghai Chest Hospital. All subjects provided written informed consents. The methods were carried out in accordance with the approved guidelines. Briefly, 5 mL of whole blood was collected from each individual and placed in a Vacutainer (BD Biosciences) without anti-coagulant. Whole blood was then left undisturbed at room temperature for 30 mins to clot. After centrifugation at 2,000 g for 10 mins at 4 ℃, the supernatant was immediately transferred into 1.5 mL Eppendorf Tubes as 0.5 mL aliquots and stored at -80 ℃. All experimental protocols were approved by Bio-X Ethics Committee of Shanghai Jiao Tong University. Strategies for cellular NABPs extraction with carbon nanomaterials Strategy A: This strategy was performed as previously described

23

. Briefly, 100 µL cell lysis

buffer (250 mM SDS, 1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF and 2% protease inhibitor Cocktail) were added to lyse ca. 5×106 cells, followed by a mechanical disruption by passing through a 200 µL pipette tip for 20 times. Cell lysate (from ca. 5×106 cells) was then mixed with 500 µg nanomaterials in 500 µL ddH2O on ice within 1 min. The composites were harvested by a 20,000 g centrifugation under 4 ℃ for 5 mins. 8


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Strategy B: 500 µg GO, cG, and cCNT was dissolved in 500 µL 250 mM SDS, respectively, to prepare cell lysis buffer mixture. Then 5×106 cells in 40 µL PBS were added slowly into cell lysis buffer with continuous mixing by the pipet tip within 30 s on the ice. The formed composites of nanomaterial with nucleic acids and NABPs were collected by a 20,000 g centrifugation (Eppendorf, Germany) under 4 ℃ for 1 min. The collected composites, either through strategy A or strategy B, were washed three times with ddH2O and then NABPs were released into nucleic acid elution buffer (8 M urea and 50 mM Tris-HCl, pH 7.4) by ultrasonication for 5 strikes (working for 2 seconds and pausing for 2 seconds) at 400 W. Cellular nucleic acids extraction efficiency To evaluate nucleic acids extraction efficiency, DNA and RNA left in the supernatant were purified by Trizol LS (Life Technologies, Carlsbad, CA). Nucleic acids from the whole cell lysates were isolated to serve as standards. Extraction was done according to the protocol provided by the vendor. Nucleic acids were quantified by NanoDrop 2000 (Thermo Scientific, Rochford, IL, USA). RNA extraction efficiency Cellular RNA was extracted by the Trizol reagent (Life Technologies, Carlsbad, CA) from 293T cells. In a typical experiment, 10 µg purified RNA was dissolved in 50 µL pure water, PBS (135 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 4.3 mM Na2HPO4, 1.4 mM K2HPO4), and

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250 mM SDS in PBS, respectively, followed by incubation with 10 µg GO, cG, G, hcCNT and cCNT for 10 mins at room temperature. After centrifugation, RNA remnant in the supernatant was measured by NanoDrop 2000. Filter-aided sample preparation (FASP) and protein digestion for Mass Spectrometry The FASP digestion was performed as previously described 30. Briefly, NABPs were precipitated with 5 volumes of 50% ethanol, 50% acetone, 0.1% acetic acid solution at -20 ℃ overnight. Protein precipitate was redissolved in 100 µL reduction solution (6 M guanidine hydrochloride in 50 mM NH4HCO3), then reduced by DTT (2 µmol), and alkylated by IAA (4 µmol). The solution was transferred to 10 kDa cutoff filter units and centrifuged at 12,000 g at 4 ℃ for 40 mins to maximal concentration. The concentrate was washed three times with 50 mM NH4HCO3 and digested for 16 hours at 37 ℃ with trypsin. Peptides were collected by centrifugation at 12,000 g at 4 ℃, and filters were washed with 50 mM NH4HCO3 to increase the yield of peptides. LC-MS/MS analysis The obtained peptides were desalted by using ZipTip C18 (Millipore, Billerica, MA), and analyzed by Thermo Scientific EASY-nLC 1000 Nano-flow UHPLC interfaced to Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher, San Jose, CA). Peptides were first loaded on a trapping column and then eluted to a 75 µm analytical column at 300 nL/min; both columns were packed with Jupiter Proteo resin (3 µm, C18, 300 Å, Phenomenex, Torrance, CA), and the length of analytical column was 15 cm. Data were acquired using HCD mode, and the mass spectrometer was operated in a data-dependent mode. MS 10


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spectra were acquired in profile mode using the Orbitrap analyzer in the m/z range between 300 and 1800 at 60,000 resolutions, and the resolution for MS/MS was set to 12500. The details for protein identification and label-free quantification are described in Supplementary methods. Bioinformatics analysis For the gene ontological (GO) analysis, the lists of proteins were submitted to the Omicsbean (http://www.omicsbean.com/) database with their UniProt accession number to analyze protein cellular component, molecular function, and biological process. For the protein-protein interaction analysis, the list of differentially expressed proteins was submitted to Ingenuity Pathway Analysis (IPA; QIAGEN, Redwood City, http://www.ingenuity.com/). Statistical Rationale The results are presented as the mean ± SD. The comparisons of quantitative data between the two groups were assessed using the two-tailed unpaired Student’s t-test, and p

Graphene Oxide Facilitated Comprehensive Analysis of Cellular

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