Mass Spectrometry Genotyping of Human Papillomavirus based on

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Biological and Medical Applications of Materials and Interfaces

Mass Spectrometry Genotyping of Human Papillomavirus based on Nano Materials High-Efficiency selective Enrichment Li Zhu, Lihui Yin, Jinjuan Xue, Zhihua Wang, and Zongxiu Nie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16694 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 11, 2018

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ACS Applied Materials & Interfaces

Mass Spectrometry Genotyping of Human Papillomavirus based on Nano Materials HighEfficiency Selective Enrichment Li Zhu†,§, Lihui Yin§, JinJuan Xue‡, Zhihua Wang*,†, Zongxiu Nie*,‡ †

State Key Laboratory of Chemical Resource Engineering, and Beijing Advanced Innovation Center for

Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 China ‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for

Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 China §

National Institutes for Food and Drug Control, Beijing 102629 China,

ABSTRACT: This work developed a novel spermine-modified nano-diamonds (SP-NDs)-based method to selectively enrich oligonucleotides for matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) analysis of human papillomavirus (HPV) virus. Our results showed that SP-NDs can effectively extract and enrich DNA oligonucleotides from sodium dodecyl sulfonate (SDS) and urea solution. In addition, SP-NDs can also selectively extract oligonucleotides from enzymes digestion products of polymerase chain reaction - restriction fragment mass polymorphism (PCR-RFMP) of HPV virus. Then the extractive can be detected by MALDI-TOF MS directly without further purification. According to the MS results, the HPV genotyping can be achieved. More importantly, with SP-NDs extraction, clinical samples infected with HPV genotype 16 and 18 can ACS Paragon Plus Environment

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be identified. The described method shows great advantages of simplicity, high selectivity and good reliability in real clinical samples. Due to improvements in DNA enrichment, extraction and purification methods, the PCR-based MALDI TOF MS for analysis of oligonucleotides maybe become more rapid, sensitive and high-throughput, is promising for analysis for DNA methylation, single nucleotide polymorphisms and other virus typing.

KEYWORDS:

spermine,

nano-diamonds,

human

papillomavirus,

enrichment,

genotyping

■ INTRODUCTION

It has been proven that continuous infection of human papillomavirus (HPV) is the primary cause of cervical cancer. So far, more than 100 different HPV genotypes have been discovered, of which about 40 have been subdivided into low-risk and high-risk types. The remaining types need further research to determine their risk degree. Low-risk HPV types may lead to genital warts while high-risk types may lead to cervical cancer. HPV 16 is by far the most carcinogenic in terms of numbers of cervical cancer cases and its precursors lesions and HPV 18 is second in terms of etiologic importance.1,2 Therefore, genotyping of HPV infection is very important for prevention and early diagnosis cervical cancer. The frequently used methods basing either on fluorescence or on mass spectrometry (MS) for genotyping are hybridization assays3,4, DNA sequencing5,6, DNA microarrays7,8 and polymerase chain reaction (PCR) assays9,10, At present, hybridization assays is considered to be an accurate method for genotyping, but they are labor intensive and unsuitable for identification of genotype variants or genotype mixtures. Besides, they are expensive due to the specific labeled probes used. So far, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-PCRrestriction fragment mass polymorphism (RFMP) has been demonstrated to be a high-throughput and economical method. MALDI-TOF MS can be applied in detection of oligonucleotides, such as DNA methylation11 and single nucleotide polymorphisms (SNP)12,13, as well as detection of alternative splicing14,15,

and

analysis ACS Paragon Plus Environment

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allele2

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specific expression16,17. However, HPV genotyping via MALDI-TOF MS RFMP analysis was limited by the tedious separation and purification by conventional slab gel electrophoresis and high performance liquid chromatography. In addition, concentration of analyte is too low to detect through MS, which may be draw wrong conclusion. PCR RFMP approaches involve buffer, enzyme and a certain amount of salts in reaction solutions, which greatly suppressed or completely eliminate the MS signal of oligonucleotides in MALDI18. MALDI-TOF MS is rapid, sensitive, and provides high accuracy without fluorescent or radioactive labeling. So it is of great significance to develop a convenient enrichment and purification oligonucleotides methods to HPV genotyping. Nanopartcles (NPs) can be applied for extraction and enrichment of target analyte from a complex matrix, including oligonucleotides19,20, small molecules21-23, peptides24-26, lysozyme27,28. Nano-diamonds (NDs) can be readily modified with diverse groups for covalent or noncovalent conjugation with biomolecules. Kong et al.29 used polylysine (PL) -coated 100 nm NDs for enrichment oligonucleotides from complex solution. But the maximal amount of the DNA oligonucleotides adsorbed to the PLcoated diamond surface at neutral pH is only 22 mg/g. Polyarginine30 has been applied to modify NDs, which can effectively extract phosphorylated peptides from complex samples. Polyarginine – coated NDs does not apply to extractive oligonucleotides. Our results show Spermine (SP) -modified NDs have more high enrichment ability of oligonucleotides than PL -coated NDs. Spermine (SP) is frequently used as stabilization helical structure, particularly in viruses31-33. In the previous literatures29,34-37, SP is rarely used to extract and enrich oligonucleotides. Here, we developed a novel SP-NDs-based method to selectively enrich and extract oligonucleotides for MALDI-TOF MS RFMP analysis of HPV virus. Studies have shown that SP-NDs can attract oligonucleotides under acidic conditions through electrostatic interaction. After treatment with SP-NDs, oligonucleotides can be extracted not only in dilute solution but also in SDS and urea solution. We also present the results of the application of SPNDs to selectively extract oligonucleotides from enzymes digestion products for HPV genotyping without purified. The results have showed that HPV 16, HPV 18 specific fragments can be extracted by SP-NDs and detected by MALDI TOF MS. More importantly, we acquired a good performance from ACS Paragon Plus Environment

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clinical sample using SP-NDs extraction. It is shown that our method simplifies the analytical procedures and identifies clinical sample. SP-NDs-based method to selectively enrich and extract oligonucleotides for MALDI-TOF MS RFMP is promising for clinical diagnosis. ■ EXPERIMENTAL SECTION

Chemicals and materials. Diamond NPs with a nominal size of 5 nm were obtained from Shenzhen Gold Diamond Abrasives Co., Ltd. Biological samples including d (TGGCATT) (7mer, mass 2111.4), d (TGTTGGGGTAAC) (12mer, mass 3716.4), d (CAACTATTTGTTA) (13mer, mass 3923.6), all PCR primers, FokI and BseGI were purchased from Thermo Fisher Co., Ltd (New Jersey, USA). Spermine (SP) and chemicals, including Acetone, sodium dodecyl sulfate (SDS), 3-hydroxypicolinic acid (3HPA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and ammonium citrate were all from SigmaAldrich (Steinheim, Germany) and used without further purification. HPV L1 (16, 18) references are provided by National Institutes for Food and Drug Control. The clinical sample was supplied by Jiangsu local Hospital. Deionized water was from a Milli Q system (Millipore, Bedford, MA, USA). Preparation of SP-NDs. The method of preparing carboxylated/oxidized diamond was based on previously described procedures38. Briefly, nano-diamond powders (5 nm) were carboxylated and oxidized in concentrated H2SO4/HNO3 (9:1 v/v ) and stirred for 3 days at 70 ℃ ， and subsequently treated in 0.1 M NaOH aqueous solution at 90 ℃ for 2 h and then in 0.1 M HCl aqueous solution at 90 ℃ for 2 h, the nano-diamonds were thoroughly washed with deionized water and collected by centrifugation, dried in vacuum at -50 ℃

for 12 h. SP-NDs were prepared via an N-(3-

dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC) mediated coupling reaction30,39. Briefly, carboxylated/oxidized diamond NPs (30 mg) were dispersed in H3BO3/NaOH buffer (5 mM, pH 5), 20 mg EDC and 60 mg SP were added in this solution. The mixture was shaken gently at room temperature for 4 h and then centrifuged at 15000 rpm for 20 min. The supernatant was removed by pipetting, and the SP-NDs precipitates were thoroughly washed with deionized water and dried in vacuum at -50 ℃ for 12 h. ACS Paragon Plus Environment

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Adsorption of Oligonucleotides. To evaluate the adsorption oligonucleotides capacity of SP-NDs, an UV-visible spectrophotometer (SHIMADZU 2600) served to determine the amounts of oligonucleotides adsorbed to SP-NDs. The sample was prepared by adding 5 μL 10 mg/mL diamond solution to 500 μL 18.4 μg/mL oligonucleotides solution in 5 mM H3BO3 buffer (adjust with 1 M HCl and 28 % ammonium hydroxide solution) at pH 2-9 in Eppendorf microcentrifuge tube. The oligonucleotide solution and the SP-NDs suspension were vortexed together in a shaker for more than 2 h to ensure equilibration, after which the mixture was centrifuged at 15000 rpm for 20 min and the supernatant was determined from the spectra acquired with a quartz cell (Hellma) of 10 mm path length at a spectrometric resolution of 2 nm. The adsorbed quantities were estimated from the changes DNA absorbance of the supernatant before and after enrichment with SP-NDs. Extraction of Oligonucleotides Using SP-NDs. Oligonucleotides solutions are prepared with 5 mM H3BO3 buffer (pH 3). Oligonucleotides mixtures are prepared by urea and 0.1% SDS with 50 nM oligonucleotides respectively. SP-NDs in deionized water were sonicated for 10 min prior to usage. The sample solutions were mixed with the SP-NDs suspensions and analyzed with MALDI-TOF MS according to the procedures described. To eliminate undesired ions, the oligonucleotide-adsorbed diamond NPs were additionally washed three times with buffer (pH 3) (200 μL in each rinse) before adding matrix solutions. MADLI-TOF MS analysis. The 3HPA was introduced as matrix. The matrix solution consisted of 15 mg of 3HPA and 2.1 mg of ammonium citrate in 500 μL of 30 % aqueous acetonitrile. 500 μL of oligonucleotides solution (pH 3) were vortexed with 1 μL 10 mg/mL diamond solution in a microcentrifuge tube for 10 min and separated by centrifugation at 15000 rpm for 20 min, supernatant was removed. 3 μL deionized water was added to wash the inner wall of the micro-centrifuge tube and mixed with centrifugation SP-NDs. 2 μL of the slurry was mixed with 2 μL of matrix solution, an aliquot (2 μL) of the mixture was deposited on the probe and air dried at room temperature. A Shimadzu AXIMA Performance MALDI-TOF instrument was operated in linear positive ion mode. The acceleration voltage was set at 20 kV. Each mass spectra was recorded with a 337 nm ACS Paragon Plus Environment

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nitrogen laser to ionize biomolecules with a value above the desorption threshold. All mass spectra were acquired by signal accumulation 100 laser shots scanned across the whole sample surface. HPV PCR-RFMP. HPV DNA was extracted from the sample, according to the manufacturers’ Instructions. HPV DNA was amplified with the consensus primers PGMY09/11 as described elsewhere 40.

The second round PCR primer pairs consisted of an sense primer specific to bases 6584–6603 (5’-

GCMCAGGGHCAYAAGGATGAATGG-3’) and an antisense primer specific to bases 6657–6626 (5’GTACTDCKDGTRGTATCHACMACGGATGTAACAAA-3’)41.The

5-nucleotide

long

sequence

(GGATG) was embedded in the primers to introduce a FokI site (a neoschizomer of BseGI) in the amplicon. Restriction enzyme digestion of PCR products was performed by mixing the PCR reaction mixtures with 10 μL of buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol and 1 unit of FokI and BseGI. The reaction mixtures were incubated at 37 ˚C for 1 h, diluted with buffer (pH 3) to 500 μL and mixed with 4 μL of the SP-NDs suspension for 10 min and washed three times with buffer (pH 3) and analyzed by MALDI following the procedures described above. ■ RESULTS AND DISCUSSION

Characterization of NDs surfaces. The Fourier-transform infrared spectroscopy (FT-IR) characterization and imaging system were performed on a PerkinElmer Spotlight 400/400N Fourier transform infrared spectrometer. Figure 1 a blue line shows the FT-IR spectrum of nano-diamonds, there is a shift to a lower wavenumber for the carboxylic group (from 1770 cm-1 to 1724 cm-1). This shift indicts the surfaces of nano-diamond have been well carboxylated/oxidized. Black line shows the FTIR spectrum of the 5 nm carboxylated/oxidized diamonds. Similar to the results reported previously42,43, a broad peak at Red line in Figure 1a displays the FT-IR spectrum of nano-diamond covalently coated with SP. The spectrum is significanly different from that of the carboxylated/oxidized nano-diamond. The prominent peaks at 2954 cm-1, 2925 cm-1 and 2858 cm-1 represent the C-H stretching vibrations of SP. We assign the most prominent peak at 1699 cm-1 to the C=O vibrations of amide bonds. The lowestACS Paragon Plus Environment

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frequency feature in the spectral region at 1129 cm-1 is most likely to derive from C-N stretching vibrations. The observations confirm the coating of SP onto the diamond nanoparticle surfaces.

Figure 1. (a) Infrared spectra of carboxylated/oxidized diamond NPs (black line) , SP-NDs (red line)and nano-diamond (blue line) (only baseline correction) (b) IR image at 1580 cm-1 wavelength of acidified diamond NPs( purple area) and SPNDs(red area) (c) the IR spectrum of extraction at point 1(black line) and 2 (red line) from IR image

Figure 1 b shows IR image at 1580 cm-1 wavelength of carboxylated/oxidized diamond NPs and SPNDs. Figure 1 c shows the spectra at point 1 and 2 of Figure 1 b. The peak at 1580 cm-1 derives from alkyl primary amide Ⅱ bond vibrations. IR image further shows spermine totally covers surfaces of diamond. Adsorption of DNA to SP- NDs Surfaces. To evaluate the adsorption efficiency of SP-NDs toward oligonucleotides, 13 mer oligonucleotide was employed as the models. Figure 2a shows the UV absorbance of the supernatant after treated with SP-NDs under different pH condition. Along with the pH value increases, the absorbance increase. Comparing with absorbance of DNA oligonucleotides without treatment, the supernatant absorbance of oligonucleotides at 260 nm after pretreated with SPNDs remarkably decreases, which means oligonucleotides are extracted to the SP-NDs. And at low pH value (PH

Mass Spectrometry Genotyping of Human Papillomavirus based on

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