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Ultra-performance liquid chromatography
Ultra-performance liquid chromatography

Journal of Chromatography B,879 (2011) 1647–1652

Contents lists available at ScienceDirect

Journal of Chromatography

B

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o m

b

Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quanti?cation of global DNA methylation in human sperms

Xiaoli Wang a ,Yongshan Suo c ,Ruichuan Yin a ,Heqing Shen b ,??,Hailin Wang a ,?

a

State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,Beijing 100085,China b

Key Lab of Urban Environment and Health,Institute of Urban Environment,Chinese Academy of Sciences,Xiamen 361021,China c

Reproduction Center of Zaozhuang Maternity and Child Care Hospital,Zaozhuang 277102,China

a r t i c l e i n f o Article history:

Received 15November 2010Accepted 3April 2011

Available online 12 April 2011Keywords:

Global DNA methylation Human sperm UPLC–MS/MS

a b s t r a c t

Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility.We developed an ultra-performance liquid chromatography/tandem mass spec-trometry (UPLC–MS/MS)method for rapid,sensitive,and speci?c detection of global DNA methylation level in human sperms.Multiple-reaction monitoring (MRM)mode was used in MS/MS detection for accurate quanti?cation of DNA methylation.The intra-day and inter-day precision values of this method were within 1.50–5.70%.By using 2-deoxyguanosine as an internal standard,UPLC–MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines.DNA methyl-transferases inhibitor 5-aza-2 -deoxycytidine can signi?cantly reduce global DNA methylation levels in treated cell lines,showing the reliability of our method.We further examined global DNA methylation levels in human sperms,and found that global methylation values varied from 3.79%to 4.65%.The aver-age global DNA methylation level of sperm samples washed only by PBS (4.03%)was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%),indicating the possible aberrant DNA methylation level in abnormal sperm cells.Clinical application of UPLC–MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.

? 2011 Elsevier B.V. All rights reserved.

1.Introduction

DNA methylation,as one of the most important epigenetic modi?cations in mammalian tissues,plays crucial roles in many biological processes such as gene transcription,genome stability,and embryogenesis [1–3].Aberrant DNA methylation,manifested as genomic DNA hypomethylation and gene-speci?c hypermethy-lation,has been found to be associated with the pathogenesis of various human diseases [4,5].Hypermethylation of tumor suppres-sor genes leads to transcriptional silencing and results in malignant transformation eventually [6,7].Genomic DNA hypomethylation,which can affect chromosome structure and activate oncogenes,has been discovered in many human cancers,such as breast can-cer,ovarian epithelial carcinoma and colorectal cancer [8–10].DNA methylation will be a useful biomarker for disease diagnosis and prognostics.

Male infertility is a common problem in today’s man health care research [11].Protamine de?ciency and oxidative DNA dam-

?Corresponding author.Tel.:+861062849600;fax:+861062849600.??Corresponding author.Tel.:+865926190771;fax:+865926190771.

E-mail addresses:hlwang@https://www.sodocs.net/doc/a614761439.html, (H.Wang),hqshen@https://www.sodocs.net/doc/a614761439.html, (H.Shen).age may be associated with infertility of some male patients,but for most patients the etiology remains unknown [12–14].Several studies implicated the possible effects of aberrant DNA methyla-tion on male infertility.Aberrant methylation of sperm DNA,such as abnormal genomic imprinting,was found in some infertile men [15,16].In mammalian sperm line,global DNA methylation level was lower than that of somatic cells,re?ecting the hypomethy-lation of satellite sequence [17,18].DNA methylation erasure and de novo DNA methylation occurs during spermatogenesis [19,20].Abnormal spermatogenesis will occur if the DNA methylation related epigenetic process is disrupted because of genetic or envi-ronmental factors [21,22].In this sense,it is expected that DNA methylation of sperm line will be a good biomarker to evaluate the fertilizing ability of spermatozoa and to study other human diseases,such as Prader–Willi syndromes [23].

Methods based on polymerase chain reaction (PCR)ampli-?cation or methylation-sensitive restriction reaction,such as methylation-speci?c PCR (MSP)[16,24],methylight [22,25]and end-labeling assay [26],were used for the analysis of locus-speci?c DNA methylation in sperms.Immunohistochemical 5-methylcytosine staining technique was applied for the measure-ment of global DNA methylation in genomic sperm DNA [14,23].However,this method is relatively non-speci?c and non-sensitive,

1570-0232/$–see front matter ? 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jchromb.2011.04.002

1648X.Wang et al./J.Chromatogr.B879 (2011) 1647–1652

so it cannot be used for precisely quantitative assessment of global DNA methylation in sperm.Other methods,such as methyl-acceptance assay(MAA)[27]and cytosine extension assay(CEA) [28],can only afford indirect information about global DNA methy-lation level,and their sensitivity and reproducibility are also not satisfactory.Therefore,it is highly desirable to develop a sen-sitive and reliable method for accurate quanti?cation of global DNA methylation in sperm.Nowadays,global methylation is usu-ally analyzed by high-performance liquid chromatography(HPLC) [29,30].However,the relatively low sensitivity,long running time and large quantities of genomic DNA required,will limit their application in large-scale clinical analysis.With the development of electrospray ionization(ESI)technique for mass spectrometry analysis(MS),liquid chromatography(LC)/MS has been used for characterization and quanti?cation of biological samples,such as nucleic acids and proteins.Friso used on-line LC/ESI-MS for the quantitative determination of global DNA methylation by measur-ing methylated cytidine residues in hydrolyzed genomic DNA using isotopically labeled internal standard[31].However,complete digestion of RNA by enzyme is required to avoid RNA contam-ination.To avoid the use of expensive isotopic label,Song and Liu developed a LC–MS/MS to analyze5-methyl-2 -deoxycytidine in enzyme-digested genomic DNA using2 -deoxyguanosine as an internal standard[32,33].Due to its high sensitivity and speci?city, LC–MS/MS will be a good tool for genomic DNA methylation anal-ysis[34,35].

In this work,we developed UPLC–MS/MS method for detec-tion of global DNA methylation in human sperms by taking advantages of ultra resolution,ultra speed and sensitivity of UPLC technology and high selectivity and speci?city of tandem MS detection.The ratio of5-methyl-2 -deoxycytidine to2 -deoxyguanosine was used for evaluation of global DNA methylation https://www.sodocs.net/doc/a614761439.html,mercial unmethylated and methylated DNA with a speci?c sequence and genomic DNA extracted from human cells treated by DNA methyltransferases inhibitor5-aza-2 -deoxycytidine were used for the con?rmation of our devel-oped method.UPLC–MS/MS technology will offer a good opportunity for the accurate quanti?cation of DNA methy-lation in human sperms,in which small variation in DNA methylation level may have important biological implications. This method will be helpful to study the epigenetic mecha-nisms of male infertility and other human genomic imprinting disorders.

2.Experimental

2.1.Reagents

5-Methyl-2 -deoxycytidine(5mdC),2 -deoxycytidine(dC),2 -deoxyguanosine(dG),thymidine(T),2 -deoxyadenosine(dA), 5-aza-2 -deoxycytidine(5-Aza-dC)and snake venom phospho-diesterase I were purchased form Sigma(St.Louis,MO,USA). Deoxyribonuclease I(DNase I),calf intestinal alkaline phosphatase (CIP)were obtained from New England BioLabs(Ipswich,MA,USA). Microcon centrifugal?lter devices were obtained from Millipore (Bedford,MA,USA).Methanol was of HPLC grade and purchased from Fisher Scienti?c(Pittsburgh,PA,USA).Ultrapure water was prepared by a Milli-Q water system(Millipore,Bedford,MA,USA). Cell-culturing reagents were obtained from Hyclone(Logan,UT, USA).

2.2.Apparatus

Nucleosides separation was achieved by Agilent1200Series Rapid Resolution LC system equipped with a vacuum degasser,binary pump SL,high performance autosampler SL with thermostat and thermostatic column compartment.A reversed-phase Zorbax SB-C182.1×100mm column(1.8-?m particle size)was used in this experiment.Agilent6410B Triple Quadrupole mass spectrom-eter(Santa Clara,CA,USA)with an electrospray ionization source was applied for mass spectrometric detection.MassHunter work-station software version B.01.03was used for data acquisition and qualitative analysis,and its version B.01.04was used for the quan-titative analysis.

2.3.Cell culture and DNA isolation

Human hepatocellular carcinoma cell line HepG2and lung adenocarcinoma cell line A549were cultured in RPMI1640 medium containing10%fetal bovine serum,100U/mL penicillin and100?g/mL streptomycin in5%CO2at37?C.Renal proximate tubular epithelial cell HK2were cultured in DMEM-F12medium, and other conditions were the same as for HepG2.HepG2cells (5×105cells)were seeded in the culture medium for24h and then treated with0.01,0.1,1and5?M of5-Aza-dC for72h.Control cells were cultured in the same way,without treatment of5-Aza-dC.Cells were harvested after72h treatment and genomic DNA was extracted using a Genomic DNA Puri?cation Kit(Promega,Madison, WI,USA),according to the manufacture’s instruction.DNA concen-tration and quality were estimated by measuring the absorbance at260nm and280nm.

2.4.Sperm collection and DNA preparation

The study was approved by local ethic committee.Semen sam-ples were collected by masturbation from24men who were undergoing evaluation for infertility at reproduction department of the hospital.The patients had been informed our research purpose and we are permitted to use the samples.After liquefaction,all the semen samples were divided into two groups.Samples from group 1were only washed by Phosphate Buffered Saline(PBS)three times at1500rpm for10min.While samples from group2were?rst cen-trifuged at1500rpm for15min using Suprasperm gradients(1ml semen,2ml40%,2ml80%)and then washed by PBS three times to remove abnormal and dead sperms.Sperm cells were collected separately for global DNA methylation analysis.

Unlike somatic cells,sperm cells have relatively compacted chromatin structures,which is tightly packaged by protamine molecules[12].Genomic DNA from sperm cells was extracted using Genomic DNA Puri?cation Kit(Promega,Madison,WI,USA), according to the manufacture’s instruction with some modi?-cations.Sperm cells and nuclei lysis solution were?rst mixed thoroughly.For complete lysis,dithiothreitol(1mol/L)and pro-teinase K(20mg/ml)were added at55?C for2h.The cell lysate was then incubated with RNase A at37?C for30min,and protein was removed by protein precipitation solution.Genomic DNA was precipitated using isopropanol and washed by70%ethanol twice. DNA concentration and purity were determined by measuring the optical density(OD)at260nm and280nm.

2.5.DNA enzymatic digestion

DNA digestion was performed as described previously[36]. Genomic DNA(1?g)extracted from human sperms or cultured cells was digested with1U DNase I,2U CIP and0.005U snake venom phosphodiesterase I at37?C for24h.Microcon centrifu-gal?lter device with a3000D cutoff membrane was used to remove protein from the digested DNA samples by centrifuging at 12,000rpm for60min.

X.Wang et al./J.Chromatogr.B879 (2011) 1647–1652

1649

Fig.1.The product ion mass spectra of5mdC.Fragment ions at m/z242.1and m/z 126were corresponding to the protonated5mdC and5-methylcytosine generated by glycosidic cleavage.

2.6.UPLC–MS/MS analysis

The mobile phase consisted of5.0%methanol and95%water (plus0.1%Formic Acid)was used for UPLC separation of the nucleosides at a?ow-rate of0.3ml/min.Enzymatically digested DNA sample(5?L each)was injected into for UPLC–MS/MS analy-sis and each run took10min.Mass spectrometry conditions were as follows:ionization mode,ESI-positive;capillary voltage,3500V; nitrogen drying gas temperature,300?C;drying gas?ow,9L/min; nebulizer,40psi.For MS/MS analysis of nucleotides,the fragmen-tor voltage was90V,collision energy was performed at5eV and scan time was100ms.Multiple-Reaction Monitoring(MRM)mode was used for the UPLC–MS/MS analysis by monitoring transition pairs of m/z242.1/126.0,m/z228.1/111.9,m/z268.1/152.0,m/z 252.1/136.0,m/z243.1/127.0,corresponding to5mdC,dC,dG,dA and dT.

3.Results and discussion

3.1.UPLC–MS/MS detection of5-methyl-2 -deoxycytidine

In our experiment,MRM mode was selected for highly sensitive quanti?cation of5-methyl-2 -deoxycytidine(5mdC).In product ion spectra of5mdC(Fig.1),m/z242.1and m/z126.0were the pre-cursor and predominant product ions of5mdC,respectively.They correspond to the protonated5mdC and5-methylcytosine pro-duced from glycosidic cleavage of5mdC.The transition pair of m/z 242.1/126.0was then used for detection of5mdC in MRM mode.

DNA sequences from5-methylcytosine DNA Standard Set(Zymo Research,Orange,CA,USA)were used for UPLC–MS/MS detection of5mdC.Cytosine DNA Standard and5-methylcytosine DNA Stan-dard are linear dsDNA which have the same897bp sequence.The only difference is that Cytosine DNA Standard contains unmodi?ed cytosines,while cytosines are fully replaced by5-methylcytosines in5-methylcytosine DNA Standard.Cytosine DNA Standard and5-methylcytosine DNA Standard were digested by DNase I,CIP,and snake venom phosphodiesterase I and the products of digestion were analyzed by UPLC–MS/MS.Chromatographic peaks of5mdC (1.9min,Fig.2A)and dC(1.4min,Fig.2B)could be clearly detected in the digested5-methylcytosine DNA Standard and Cytosine DNA Standard,respectively.Transition pairs of m/z268.1/152.0, 252.1/136.0,243.1/127.0corresponding to dG,dA and dT were monitored at the same time.Surprisingly,the area under the

dT Fig.2.UPLC–MS/MS chromatograms of DNA hydrolysate from5-methylcytosine DNA standard(A)and cytosine DNA standard(B).5mdC(1.9min)and dC(1.4min) were detected by monitoring m/z242.1/126and228.2/111.9,respectively.

peak is much larger than the area under the dA peak although they have same concentration.This difference may be attributed to the different protonation ef?ciency of these nucleosides in MS/MS detection.We speculated that it was dif?cult for thymi-dine to be protonated,so T peak was relative small.For adenosine, it is supposed to be converted to inosine by a small amount of adenosine deaminase during the process of enzymatic https://www.sodocs.net/doc/a614761439.html,plete separation of5mdC from other nucleosides by UPLC could be achieved within10min.Potential interference from other nucleotides was not observed due to the complete separation of these nucleotides,showing high speci?city of our method.

3.2.Quanti?cation of global DNA methylation level

To quantify genomic DNA methylation level,the ratio of5-methyl-2 -deoxycytidine to2 -deoxyguanosine was estimated,in which dG was chosen as an internal standard.The use of expen-sive isotope labeled standards could be avoided and the in?uence of insuf?cient DNA hydrolysis could be corrected in this way.Serial dilutions of5mdC to dG were prepared and the ratios of5mdC to dG were chosen at0.1%,0.5%,1%,2.5%,5%,and10%because global DNA methylation levels were typically varied from2%to7%in mam-malians.Calibration curve was obtained by plotting the ratio of MRM/MS signal of5mdC to dG against[5mdC]/[dG](R2=0.999)

1650X.Wang et al./J.Chromatogr.B 879 (2011) 1647–1652

Table 1

Precision of UPLC–MS/MS method for the determination of 5mdC (n =3).DNA sample

Intra-day Inter-day Ave.+S.D.

C.V.%Ave.+S.

D. C.V.%[5mdC]/[dG](%)1 1.03±0.015 1.50 1.02±0.042 4.085 5.17±0.097 1.87 5.14±0.160 3.1210

9.90±0.167 1.699.74±0.272 2.80Genomic DNA A549 4.13±0.134 3.24 4.28±0.244 5.70HepG2 3.68±0.141 3.83 3.81±0.161 4.24HK2

5.13±0.099

1.94

5.39±0.307

5.70

and used for the following estimation of global DNA methylation level.

Global DNA methylation levels in three cultured cell lines (HepG2,A549and HK2)were evaluated by UPLC–MS/MS https://www.sodocs.net/doc/a614761439.html,ing the ratio of 5mdC to dG as the indicator of global DNA methy-lation level,the corresponding average values for HepG2,A549,and HK2were 3.81%,4.28%,and 5.39%as indicated in Table 1.It was evident that the global DNA methylation levels of HepG2and A549were obviously lower than that of HK2.It was well known that HepG2and A549were cancer cell lines,while HK2was an immortalized proximal tubule epithelial cell line from normal adult human kidney.This was consistent with the fact that DNA methyla-tion levels of tumor-derived genomic DNA were signi?cantly lower than that in normal cell lines [37].The applicability of the devel-oped UPLC–MS/MS method for global DNA methylation detection was con?rmed.

To further test the applicability of UPLC–MS/MS method to DNA methylation detection,genomic DNA hypomethylation caused by 5-Aza-dC,a representative inhibitor of DNA methyltransferase,was assessed.HepG2cells were treated by 5-Aza-dC (0,0.01,0.1,1and 5?M)for 72h.Genomic DNA was extracted from harvested cells and then enzymatically digested for UPLC–MS/MS detection.Global DNA methylation level decreased with the increasing concentra-tion of 5-Aza-dC.Even treated with 5-AzadC as low as 0.01?M,the inhibition of global DNA methylation (3.6%)could be observed obvi-ously (Fig.3).This result demonstrated that UPLC–MS/MS method was applicable to the accurate evaluation of small changes in global DNA methylation

level.

Fig.3.In?uence of 5-Aza-dC on the global DNA methylation level of HepG2cells.Reduction of global DNA methylation level could be detected after the cells being treated with 0.01?M of 5-Aza-dC.Treated with 5?M of 5-Aza-dC for 72h,the global DNA methylation level of HepG2decreased about

40%.

Fig.4.The change in the MS signal ratio of 5mdC to dG by varied DNA content from 5to 500ng (A)and the linear relationship between the mass signal area of 5mdC and DNA contents (B).

3.3.Validation of UPLC–MS/MS method

For UPLC–MS/MS method validation,intra-day and inter-day precision were evaluated for the ratio of 5mdC to dG at 1%,5%and 10%,and the results were summarized in Table 1.The intra-day pre-cision values showed as covariance (C.V.)varied from 1.50%to 1.87%and the inter-day precision values ranged from 2.80%to 4.08%.For global DNA methylation detection of cultured cell lines,the intra-day precision values ranged from 1.94%to 3.83%and the inter-day precision values varied from 4.24%to 5.70%.All these values were within the accepted guidance for industry (bioanalytical method validation),showing little variability and good reproducibility of the UPLC–MS/MS method.

Detection limit of UPLC–MS/MS method was also estimated using a series of diluted A549genomic DNA (5ng,20ng,50ng,100ng,and 500ng).There was a linear relationship (R 2=0.99)between the mass signal area of 5mdC and DNA content ana-lyzed (Fig.4B),while the ratios of mass signal area of 5mdC to dG remained constant (C.V.8.15%)even when the amount of DNA assayed varied from 20ng to 500ng (Fig.4A).Based on the UPLC–MS/MS results,with 5ng genomic DNA analyzed,it still could be detected when the global DNA methylation level was as low as 0.16%(S/N =3).Therefore,by taking advantage of the low DNA con-sumption and high sensitivity,UPLC–MS/MS method could be used for global DNA methylation analysis.

X.Wang et al./J.Chromatogr.B879 (2011) 1647–1652

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Fig.5.UPLC–MS/MS chromatogram of DNA hydrolysate from human sperms(A) and the global DNA methylation levels in human sperms(B).Sperm samples of group 1were only washed by PBS,while abnormal and dead sperm cells were removed by density gradient centrifugation from samples of group2.

3.4.Analysis of global DNA methylation level in human sperm samples

With the con?rmation of UPLC–MS/MS method described above,global DNA methylation level in human sperm samples was detected.Semen samples were divided into two groups according to different pre-treatment protocols.Genomic DNA extracted from sperm cells was digested to nucleosides using DNase I,CIP and snake venom phosphodiesterase I,and the digestion products were assayed using UPLC–MS/MS method.5mdC(1.9min)and dC(1.4min)were detected in the UPLC–MS/MS chromatogram of genomic DNA extracted from human sperm cells,with complete separation of all nucleotides assayed(Fig.5A).

By calculating the ratio of5mdC to dG,global DNA methylation level of detected human sperm ranged from3.79%to4.65%(Fig.5B). Comparing the results from sperm of group1with that of group 2,average global DNA methylation level of group1(4.03±0.15%) was relatively lower than that of group2(4.25±0.18%).In group 2,abnormal and dead sperm cells were removed by density gradi-ent centrifugation,so we speculated that the lower average global DNA methylation level of group1may be associated with the exis-tence of abnormal and dead sperm cells.Aberrant DNA methylation reprogramming may occur during spermatogenesis in abnormal and dead sperm cells.From the preliminary result,we considered that pre-treatment of semen samples might in?uence the detec-tion of global DNA methylation level and aberrant DNA methylation may partly contribute to abnormal spermatogenesis and compro-mised sperm function.The present result need to be tested in a larger cohort/population.

4.Conclusion

In summary,we describe a UPLC–MS/MS method for detection of global DNA methylation levels in human sperm https://www.sodocs.net/doc/a614761439.html,pared with conventional methods used for global DNA methylation detec-tion of human sperms,the ultra resolution,high sensitivity and speci?city of UPLC–MS/MS method make it a very useful tool for global DNA methylation assay.With the accurate quanti?cation of sperm DNA methylation level using UPLC–MS/MS,DNA methyla-tion could be used as a good biomarker for clinical evaluation of human sperm quality and elucidation of epigenetic decisions in mammalian germ cells.

Acknowledgments

The work was supported by the grants from the National Basic Research Program of China(2007CB407305,2009CB421605, and2010CB933502)and the National Natural Science Founda-tion of China(21077129,20921063,20877091,20890112,and 20737003)to Dr.H.Wang,and the grants from the Chinese Academy of Sciences(CAS)100Talents Program,CAS/SAFEA Inter-national Partnership Program(KZCX2-YW-T08)and Ministry of Science and Technology of the People’s Republic of China for Com-bined Pollution and Ecosystem Health in Urban Agglomeration (2009DFB90120)to Dr.Shen.We thank all the people who con-tributed their samples and work in this study.

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盘点虎牙直播实力主播 首位叫板王者

盘点虎牙直播实力主播首位叫板王者荣耀职业战队王者荣耀有多火就意味着王者荣耀的直播有多受欢迎。除了玩家们耳熟能详的嗨氏、裴小峰之外,虎牙直播还有一群犀利无比的王者荣耀主播,如果将这些主播凑成一个队伍,他们的阵容实力根本不虚职业队伍。究竟这些能够媲美职业选手的主播们是何方神圣,让我们一起来看下吧。 实力貂蝉:九日 作为虎牙直播的实力主播,九日的貂蝉和嗨氏同属于国服最强貂蝉系列。嗨氏的貂蝉更追求的是一张循序渐进的打法,而九日的貂蝉却往往喜欢在刀尖上跳舞,团战中锁定对方主力,在力求追杀对方伤害来源的情况下闪转腾挪,保证了貂蝉的存活率。这位百星王者的不知火舞也是一样的飘逸,操作与技术着实让人折服。除了直播中的实力,九日还在仙阁夺冠后的仙阁挑战赛中打败过仙阁的主力小羽,一时之间传为佳话。 死神露娜:曹操大表哥 虽然名为曹操大表哥,但大表哥最让人记住的英雄却是露娜。众所周知,由于露娜技能设定的原因,露娜一般选择的只有一去无归的刺客路线,但在曹操的手下,露娜的生存能力大大的得到 拓展,大招几近无限刷新的流氓打法也让他的貂蝉在钻石王者领域横冲霸道,喜欢露娜的玩家们可以观摩下这位主播的操作。

灭世孙尚香:李太白 除了本命英雄李白的神级实力,作为虎牙第一孙尚香,李太白对于孙尚香的了解超出常人。孙尚香机动性强且在团战中总能打出爆发伤害。但孙尚香也存在着容易被敌方突进和血量较低的缺点,李太白依靠一技能翻滚突袭的风骚走位经常在团战中上演极限反杀的表现。同时,由于自己打出来成吨的伤害,这位胖子主播也成功和李白的潇洒绝缘,给玩家们带来各种杀伐果决的操作后,继续搜寻着下一个猎物。 双英雄制霸:成成 在别的主播擅长的大多是一个英雄的时候,成成的嬴政和赵云给足了玩家们看点。曾经的虎牙第一猴王早已经不再满足于大闹天空,不管是前期给力的赵云还是后期发威的嬴政,成成能否信手拈来。作为王者荣耀明星导师的成成在直播时总会交给玩家们一些骚套路,这也是为什么他的赵云总能七进七出不死嬴政暗处放大豪取五杀。

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