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Showcasing research from Ying-Lin Zhou and Xin-Xiang Zhang’s

group, College of Chemistry and Molecular Engineering,

Peking University, Beijing, China.

Ultrasensitive and simultaneous determination of RNA

modified nucleotides by sheathless interfaced capillary

electrophoresis–tandem mass spectrometry

A label-free ultrasensitive method was established for the

simultaneous determination of RNA modified nucleotides

based on a sheathless capillary electrophoresis–tandem

mass spectrometry system, which could be used to

quantify the RNA modifications in rare samples.

As featured in:

ISSN 1359-7345

COMMUNICATION Sandrine Dourdain et al . Probing the existence of uranyl trisulfate structures in the AMEX solvent extraction process

ChemCommChemical Communicationsrsc.li/chemcomm

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See Ying-Lin Zhou, Xin-Xiang Zhang et al ., Chem . Commun ., 2019, 55 , 7595.

This journal is©The Royal Society of Chemistry 2019 Chem. Commun., 2019, 55, 7595--7598 | 7595

Cite this:Chem. Commun., 2019,

55, 7595

Ultrasensitive and simultaneous determinationof RNA modified nucleotides by sheathlessinterfaced capillary electrophoresis–tandem massspectrometry†

Yue Yu,a Si-Hao Zhu,b Fang Yuan, a Xiao-Hui Zhang,c Yan-Ye Lu,b

Ying-Lin Zhou *a and Xin-Xiang Zhang*a

A label-free ultrasensitive determination of eight RNA modified

nucleotides simultaneously was first established based on a sheath-

less capillary electrophoresis–tandem mass spectrometry system.

This system performed well using only 500 pg–5 ng practical RNA

samples, and a downward trend of most target nucleotides in HCT

116 cells was observed with the increase of nickel concentration.

In addition to canonical A, C, G, and U residues, decades ofresearch have identified more than 100 different types of post-transcriptional RNA modifications in the three kingdoms oflife.1 These modifications are present in abundant noncodingRNAs (ncRNAs), which are important for the functions of thesencRNAs in translation and splicing.2,3 Messenger RNA (mRNA)and long noncoding RNA (lncRNA) have also demonstrated thepresence of modifications, such as the 50 cap and RNA editing.4–6

In particular, recent studies have proved that several modifica-tions, including N6-methyladenosine (m6A), N1-methyladenosine(m1A), and pseudouridine (c), are found in eukaryotic mRNA andcan influence the metabolism and functions of mRNA.1 Forexample, m6A can regulate the pluripotency of embryonic stem cells(ESCs) by affecting the mRNA levels and stability of pluripotencygenes.7 Carlile et al. found that most of the c modifications onmRNA were related to the response of cells to environmentalsignals.8 Thus, ‘RNA epigenetics’ was first defined in 2010,forming epigenetics with the modifications to DNA and histonetogether.9 So far, several RNA modifications have been implicatedin human disease, while others’ functions are still unknown.10–12

There’s no doubt that they are closely related to human lifeactivities, needing more research studies in the future.

To date, increasing evidence has demonstrated that toxicheavy metals, such as arsenic (As), cadmium (Cd), chromium(Cr) and nickel (Ni), affect epigenetic alterations.13–15 Ni is anon-essential metal of great environmental concern because itis widely used in industrial and medical processes. Excessiveexposure to Ni2+ has adverse effects on human health.16 Due toits weak mutagenesis, epigenetic changes have been implicatedin the mechanism of Ni-induced health effects, suggesting thatthe influence of Ni2+ on an organism at the molecular level canextend well beyond interactions with the DNA sequence.17 Asearly as 1995, Lee et al. found that exposure of cells to Ni causedchanges in DNA methylation, which led to the inactivation ofgene expression.18 Then, changes in DNA methylation followingexposure to Ni2+ were also observed in vivo.15 In addition to DNAmethylation, other Ni-induced epigenetic changes were shown tocause global histone modifications, such as increases in H3K9dimethylation.19 However, there are few studies on the effects ofexposure to Ni2+ on RNA epigenetics, and the changes and themechanism are largely unknown.

Although research studies into RNA modifications progressat a high rate, it is quite a challenge to quantify these RNAmodifications, because of their low abundance, as well as theinterference from bulky normal nucleosides. Many analyticalmethods have been developed over the past decades to determinethe content of modifications, such as thin layer chromatography(TLC),20 gas chromatography (GC),21,22 liquid chromatography(LC),23,24 capillary electrophoresis (CE)25,26 and various sequen-cing technologies.27–29 Among these methods, high performanceliquid chromatography coupled with mass spectrometry (HPLC-MS)30–32 is well-recognized as a conventional bioanalytical plat-form for quantification and validation of various modifications.With the use of tandem mass spectrometry (MS/MS), as well asthe multiple reaction monitoring (MRM) mode, the sensitivitiesof modifications have been further improved.33 However, thesample consumption of several hundred nanograms is still toolarge for analysis of some biological samples, and the constraintthat proteins normally cannot be injected into a conventionalreversed-phase chromatographic column makes it necessary for

a Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory

of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking

University, Beijing 100871, China. E-mail: zhouyl@pku.edu.cn, zxx@pku.edu.cn;

Fax: +86-10-62754112; Tel: +86-10-62754112b Molecular Imaging Lab, Department of Biomedical Engineering, Peking University,

Beijing 100871, Chinac State Key Laboratory of Natural and Biomimetic Drugs, Peking University,

38 Xueyuan Road, Beijing 100191, China

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc03195b

Received 25th April 2019,Accepted 28th May 2019

DOI: 10.1039/c9cc03195b

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7596 | Chem. Commun., 2019, 55, 7595--7598 This journal is©The Royal Society of Chemistry 2019

samples to be pre-treated after enzymatic hydrolysis, which resultsin an unnecessary loss of samples and affects the further devel-opment of research in the field of RNA epigenetic modifications.

Capillary electrophoresis–tandem mass spectrometry (CE-MS/MS) is a powerful alternative to HPLC-MS/MS. Due to theinherent advantages of CE including high separation efficiency,diverse separation modes for different types of samples and lowsample consumption, the performance of CE-MS/MS in analysis ofsmall amounts of biological samples is more prominent. Moreover,a sheathless interface coupled with a porous tip sprayer designedfor CE-MS coupling can solve the problem of dilution, greatlyincreasing the detection sensitivity, and meet the requirementsof biochemical analysis.34,35 Our lab successfully combined CEwith triple quadrupole mass spectrometry through a NanoSprayIII source. By optimizing the position of the sprayer and otherdetection conditions, stable and sensitive MS signals wereobtained, which provided technical support for the implementa-tion of CE-MS/MS.

In this study, a label-free ultrasensitive method for thesimultaneous determination of RNA modified nucleotideswas first established based on a sheathless CE-MS/MS systemwith the MRM mode (Scheme S1, ESI†). The perfect perfor-mance of this system was demonstrated to identify and quantifyeight important and functional RNA modifications, includingm6A, m1A, 20-O-methyladenosine (Am), 5-methylcytidine (m5C),20-O-methylcytidine (Cm), 20-O-methylguanosine (Gm), c and20-O-methyluridine (Um). With the parameters optimized, all theseeight RNA modifications could be easily detected by using about500 pg total RNA samples and 5 ng mRNA samples without anysample pretreatment steps, derivation steps or signal enhancingmethods, which was the most sensitive method compared to otherreported methods.24,33,36 In addition, the determination of theeight RNA modifications in 100 HeLa cells was achieved using thismethod. The effects of exposure to Ni2+ on RNA epigenetics werefurther investigated using this method.

To validate the feasibility of CE-MS/MS for the determinationof RNA modifications, four normal nucleosides (A, C, G and U)and standards of the eight modified nucleosides were mixedtogether as standard solutions (the concentration of each stan-dard is 50 nM) for analysis. A bare fused silica capillary with thesimplest capillary zone electrophoresis (CZE) mode was used forCE separation. 10% acetic acid solution (pH 2.2) was chosen asboth the background electrolyte (BGE) and conductive liquid.ESI was performed in positive ionization mode, and the targetanalytes were monitored in MRM mode. The MRM parametersof all nucleosides were optimized to achieve maximal detectionsensitivity (Table S1, ESI†). As shown in Fig. 1, all nucleosideswere detected within 15 min under these simple separationconditions with a good signal to noise ratio.

To check the performance of the method for the determina-tion of modifications, linear dynamic ranges, limits of detec-tion (LODs) and precision were evaluated. The linearity of themethod was investigated using twelve nucleoside standards.The calibration curves of the eight modified nucleosides wereconstructed by plotting the modified nucleoside/correspondingnormal nucleoside peak area ratios (such as m6A/A) versus their

actual molar ratios. The linear dynamic ranges, linear equa-tions and LODs of the eight modified nucleosides are shown inTable 1, and the calibration curves are shown in Fig. S1 (ESI†).According to the results shown in Table 1, excellent linearity forthe eight modified nucleosides was achieved. The LODs (S/N = 3)obtained by continuous dilution ranged from 25 pM (2.5 amol in100 nL) to 2 nM (200 amol in 100 nL). The sensitivity of thismethod is more than 10 times higher than those of reportedHPLC-MS/MS assays (Table S2, ESI†).33,36 Each standard samplewas analyzed three times so that the repeatability of the methodcould also be examined. The RSD for migration time was lessthan 4% (not shown), while the RSD for the peak area ratio was lessthan 10% (Fig. S1, ESI†). The performance of this method wasfurther evaluated by determining the intra-day and inter-day preci-sion of the 50 nM A standard sample. The intra-day RSD and theinter-day RSD for the peak area were both less than 10%, demon-strating that good reproducibility was achieved by CE-MS/MS.

These results showed that the modified nucleosides could bewell detected by the simplest CE mode and as low as amol LODswere easily achieved. The ultrahigh sensitivity obtained by thissheathless CE-MS/MS system should be attributed to two reasons.Firstly, the porous tip sprayer for sheathless CE-MS avoided thesolvent dilution effect, the hydrodynamic laminar flow effect andthe post-capillary dilution effect. Secondly, using MS/MS as well

Fig. 1 MRM chromatograms of a mixture of twelve nucleosides (50 nM)obtained by sheathless CE-MS/MS using a porous tip sprayer. The legendsin the figure are arranged in the order of the peaks of the nucleosides. Theretention time for each nucleoside is shown in Table S1 (ESI†). SheathlessCE-MS/MS conditions: capillary electrophoresis, bare fused silica capillarywith a porous tip, total length 100 cm 30 mm i.d. 150 mm o.d.; voltage,25 kV; current, 3.2 mA; BGE and conductive liquid, 10% acetic acid;injection sequence, 2.5 psi 60 s sample, 2.5 psi 5 s BGE; mass spectro-metry, spray voltage, 1750 V.

Table 1 Linear ranges, linear equations and LODs of RNA modifications

Linear range (%) Linear equation R2 LODs (nM)

m1A 0.01–5 y = 2.29 � 10�2x � 2.80 � 10�4 0.999 0.050m6A 0.01–5 y = 1.78 � 10�2x + 1.43 � 10�5 0.999 0.050Am 0.01–10 y = 1.35 � 10�2x + 3.21 � 10�3 0.989 0.025m5C 0.01–10 y = 1.38 � 10�2x + 1.78 � 10�5 0.995 0.025Cm 0.01–10 y = 1.37 � 10�2x + 6.86 � 10�4 0.999 0.025Gm 0.01–25 y = 1.02 � 10�2x � 8.89 � 10�4 0.998 0.050Um 0.01–25 y = 1.10 � 10�2x + 1.24 � 10�4 0.997 2.000c 0.01–25 y = 1.59 � 10�3x + 1.38 � 10�4 0.996 2.000

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as the MRM mode can further improve the sensitivity, because ofthe double screening of multiple target ions, eliminating theinterference of a large number of background ions. With all thesetogether, sheathless CE-MS/MS is a great method that can beused to simultaneously determine various modified nucleosideswith simple sample preparation steps and can achieve extremelylow LODs.

We further investigated the potential of the sheathlessCE-MS/MS system for the identification and quantification ofmodified nucleosides in RNA extracted from practical biologi-cal samples. We first experimented with the mRNA extractedfrom HeLa cells. About 5 ng mRNA was digested, and the finalsolution was then analyzed using sheathless CE-MS/MS directly(Fig. S2, ESI†). The results perfectly matched with the statisticsobtained by LC-MS/MS, which used 300 ng RNA samples (Table S3,ESI†), indicating that our methods had high sensitivity, excellentaccuracy and an excellent quantification ability. In addition,three independent treatments of RNA samples over a day andfor three consecutive days gave intra-day and inter-day RSDs. Theresults showed that the intra- and inter-day RSDs were less than10% and 15%, respectively (Table S4, ESI†), demonstrating thegood reproducibility of our method dealing with the practicalsample. We further achieved the determination of the eightmodifications in 100 HeLa cells (Fig. S3, ESI†), demonstratingthe feasibility of applying this method to the detection ofprecious samples such as embryo samples.

Then, 500 pg total RNA and 5 ng mRNA samples extractedfrom HCT 116 cells, whose amounts were too small to beanalyzed by LC-MS/MS, were analyzed by CE-MS/MS (Fig. S4,ESI†). These HCT 116 cells were treated with different concen-trations of Ni2+. With the increase of Ni2+ concentration, thesurvival rate of cells decreased, and the contents of modifiednucleosides showed different trends, which initially indicated thatnickel had potential effects on cells’ vital activity by affecting thelevel of methylation of nucleic acids. The trends of m6A in totalRNA and mRNA are shown in Fig. 2, and those of others areshown in Fig. S5 and S6 (ESI†). Based on the results, we had somespeculations. First, the content of m6A decreased in total RNA andmRNA (Fig. 2). It was reported that as the proportion of m6Adecreases, the differentiation ability of cells decreases and theproliferation ability increases.37 The trends of m6A suggested thatsurviving cells exposed to Ni2+ might have a stronger proliferationability, which might lead to carcinogenesis or tumor metastasis.Secondly, Am, Cm, Gm and Um showed downward trends in totalRNA, but remained basically unchanged in mRNA, suggesting that

the effects of Ni2+ on Am, Cm, Gm and Um might be regulated byncRNA. However, c did not change significantly in total RNA, butdecreased in mRNA, which suggested that c in mRNA might berelated to cell responses to environmental signals such as Ni2+

treatment. The relevant mechanism of this phenomenon will bestudied in our further work with the combination of variousmethods.

In conclusion, we developed a sheathless CE-MS/MS methodthat could simultaneously identify and quantitate eight modifiednucleosides in small amounts of RNA samples without any deriva-tion steps or signal enhancing methods. This method presented agood separation resolution of the nucleosides involved andachieved extremely low LODs of these special nucleosides. Excellentlinearity and satisfactory reproducibility of the method were alsodemonstrated. Using this method, we successfully quantified thesemodifications in HeLa cell mRNA as well as in total RNA andmRNA extracted from HCT 116 cells treated with different concen-trations of Ni2+. The results show that this method is simple andsensitive enough to be used for detecting and quantifying the RNAmodifications in rare samples, providing an opportunity forscientists to monitor biology processes that could not be inves-tigated before. Moreover, the changing trends of Ni2+ treatmentwere also interesting enough to attract further research.

This work was supported by the National Natural ScienceFoundation of China (no. 21575005 and 21775006).

Conflicts of interest

There are no conflicts to declare.

Notes and references1 M. A. Machnicka, K. Milanowska, O. Osman Oglou, E. Purta,

M. Kurkowska, A. Olchowik, W. Januszewski, S. Kalinowski,S. Dunin-Horkawicz, K. M. Rother, M. Helm, J. M. Bujnicki andH. Grosjean, Nucleic Acids Res., 2013, 41, D262–D267.

2 P. F. Agris, Nucleic Acids Res., 2004, 32, 223–238.3 J. Karijolich, C. Yi and Y. T. Yu, Nat. Rev. Mol. Cell Biol., 2015, 16,

581–585.4 I. Topisirovic, Y. V. Svitkin, N. Sonenberg and A. J. Shatkin, Wiley

Interdiscip. Rev.: RNA, 2011, 2, 277–298.5 N. Fossat, K. Tourle, T. Radziewic, K. Barratt, D. Liebhold,

J. B. Studdert, M. Power, V. Jones, D. A. Loebel and P. P. Tam, EMBORep., 2014, 15, 903–910.

6 K. Nishikura, Nat. Rev. Mol. Cell Biol., 2016, 17, 83–96.7 S. Geula, S. Moshitch-Moshkovitz, D. Dominissini, A. A. Mansour,

N. Kol, M. Salmon-Divon, V. Hershkovitz, E. Peer, N. Mor,Y. S. Manor, M. S. Ben-Haim, E. Eyal, S. Yunger, Y. Pinto,D. A. Jaitin, S. Viukov, Y. Rais, V. Krupalnik, E. Chomsky,M. Zerbib, I. Maza, Y. Rechavi, R. Massarwa, S. Hanna, I. Amit,E. Y. Levanon, N. Amariglio, N. Stern-Ginossar, N. Novershtern,G. Rechavi and J. H. Hanna, Science, 2015, 347, 1002–1006.

8 T. M. Carlile, M. F. Rojas-Duran, B. Zinshteyn, H. Shin, K. M. Bartoliand W. V. Gilbert, Nature, 2014, 515, 143–146.

9 C. He, Nat. Chem. Biol., 2010, 6, 863–865.10 A. Klungland and J. A. Dahl, Curr. Opin. Genet. Dev., 2014, 26, 47–52.11 G. Lichinchi, S. Gao, Y. Saletore, G. M. Gonzalez, V. Bansal, Y. Wang,

C. E. Mason and T. M. Rana, Nat. Microbiol., 2016, 1, 16011.12 M. Helm and J. D. Alfonzo, Chem. Biol., 2014, 21, 174–185.13 S. Chanda, U. B. Dasgupta, D. Guhamazumder, M. Gupta,

U. Chaudhuri, S. Lahiri, S. Das, N. Ghosh and D. Chatterjee, Toxicol.Sci, 2006, 89, 431–437.

14 T. Doi, P. Puri, A. McCann, J. Bannigan and J. Thompson, Toxicol.Sci, 2011, 120, 475–480.

Fig. 2 The trends of m6A with the increase of nickel concentration in(A) total RNA and (B) mRNA. The error bars represent the SDs of threeindependent analyses. *p o 0.1; **p o 0.05; ***p o 0.01.

ChemComm Communication

Publ

ishe

d on

30

May

201

9. D

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ing

Uni

vers

ity o

n 8/

13/2

019

4:20

:17

AM

. View Article Online

7598 | Chem. Commun., 2019, 55, 7595--7598 This journal is©The Royal Society of Chemistry 2019

15 K. Salnikow and A. Zhitkovich, Chem. Res. Toxicol., 2008, 21, 28–44.16 K. K. Das, S. N. Das and S. A. Dhundasi, Indian J. Med. Res., 2008,

128, 412–425.17 J. E. Sutherland and M. Costa, Ann. N. Y. Acad. Sci., 2003, 983, 151–160.18 Y. W. Lee, C. B. Klein, B. Kargacin, K. Salnikow, J. Kitahara,

K. Dowjat, A. Zhitkovich, N. T. Christie and M. Costa, Mol. Cell.Biol., 1995, 15, 2547–2557.

19 H. Chen, Q. Ke, T. Kluz, Y. Yan and M. Costa, Mol. Cell. Biol., 2006,26, 3728–3737.

20 H. Grosjean, L. Droogmans, M. Roovers and G. Keith, MethodsEnzymol., 2007, 425, 55–101.

21 C. W. Gehrke and C. D. Ruyle, J. Chromatogr. A, 1968, 38, 473–491.22 D. B. Lakings and C. W. Gehrke, J. Chromatogr. A, 1971, 62, 347–367.23 C. W. Gehrke and K. C. Kuo, J. Chromatogr. A, 1989, 471, 3–36.24 J. Xu, A. Y. Gu, N. R. Thumati and J. M. Y. Wong, Genes, 2017, 8, 219.25 H. M. Liebich, G. Xu, C. Di Stefano and R. Lehmann, J. Chromatogr.

A, 1998, 793, 341–347.26 M. G. Cornelius and H. H. Schmeiser, Electrophoresis, 2007, 28,

3901–3907.27 M. Schaefer, T. Pollex, K. Hanna and F. Lyko, Nucleic Acids Res.,

2009, 37, e12.

28 V. de Crecy-Lagard, S. Edelheit, S. Schwartz, M. R. Mumbach,O. Wurtzel and R. Sorek, PLoS Genet., 2013, 9, e1003602.

29 U. Birkedal, M. Christensen-Dalsgaard, N. Krogh, R. Sabarinathan,J. Gorodkin and H. Nielsen, Angew. Chem., Int. Ed., 2015, 54, 451–455.

30 D. Su, C. T. Chan, C. Gu, K. S. Lim, Y. H. Chionh, M. E. McBee,B. S. Russell, I. R. Babu, T. J. Begley and P. C. Dedon, Nat. Protoc.,2014, 9, 828–841.

31 M. Taoka, Y. Nobe, M. Hori, A. Takeuchi, S. Masaki, Y. Yamauchi,H. Nakayama, N. Takahashi and T. Isobe, Nucleic Acids Res., 2015,43, e115.

32 S. C. Pomerantz and J. A. McCloskey, Methods in Enzymology,Academic Press, 1990, vol. 193, pp. 796–824.

33 B. F. Yuan, Methods Mol. Biol., 2017, 1562, 33–42.34 M. Moini, Anal. Chem., 2007, 79, 4241–4246.35 R. Haselberg, C. K. Ratnayake, G. J. de Jong and G. W. Somsen,

J. Chromatogr. A, 2010, 1217, 7605–7611.36 W. Huang, M. D. Lan, C. B. Qi, S. J. Zheng, S. Z. Wei, B. F. Yuan and

Y. Q. Feng, Chem. Sci., 2016, 7, 5495–5502.37 C. Zhang, D. Samanta, H. Lu, J. W. Bullen, H. Zhang, I. Chen, X. He

and G. L. Semenza, Proc. Natl. Acad. Sci. U. S. A., 2016, 113,E2047–E2056.

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