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Determination of Nucleosides in Escherichia coli by Rapid Resolution Liquid Chromatography–Tandem Quadrupole Mass Spectrometry
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 41, Issue 1, January 2013 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2013, 41(1), 36–41.
RESEARCH PAPER
Determination of Nucleosides in Escherichia coli by Rapid Resolution Liquid Chromatography-Tandem Quadrupole Mass Spectrometry XIE Yu-Ping1,2, TIAN Jing1,*, GAO Peng2, XU GUO-Wang2, FEI Xu1, WANG Yi1 1 2
School of Bioengineering, Dalian Polytechnic University, Dalian 116034, China Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Abstract: A sensitive and selective method was developed for the quantitative determination of nucleosides in Escherichia coli. An Agilent 1290 HPLC was used with mobile phase A (10 mM ammonium acetate) and mobile phase B (acetonitrile) at a flow-rate of 0.2 mL min–1 coupled with a mass spectrometry scanned in multiple reaction monitoring (MRM) mode. In the result, the limit of quantification and detection of 19 nucleosides were in the range of 0.05–20 µg L–1 and 0.02–10 µg L–1. The linearity of the detected nucleosides was excellent with R2 > 0.99 and the limits of detection were all satisfied. The recoveries for the compounds were between 79.0%–119.8%, and the relative standard deviation was below 14%. The method was capable of quantitation 7 nucleosides in Escherichia coli, of which guanosine was found to decrease along with culture phases. Key Words: Rapid resolution liquid chromatography-tandem mass spectrometry; Nucleoside; Escherichia Coli
1
Introduction
Nucleic acids that exist in living cells include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and the latter mainly exists in the form of ribosomal RNA, messenger RNA and transfer RNA(tRNA). Besides four normal bases, there are also some minor bases in tRNA, which are usually generated by chemical modification of the four major ones during post-transcription or in exceptional cases. Decades of studies have revealed mainly about 100 ribonucleoside structures incorporated as post-transcriptional modifications across all organisms and more than 40 in the prokaryotes[1,2]. The effect of modified nucleosides on RNA function is still unknown. In general, tRNA modifications enhance ribosome binding affinity, reduce misreading and modulate frame-shifting, all of which affect the rate and
fidelity of translation[3–5]. Recent studies suggested that modified nucleosides could improve the survival ability of Saccharomyces cerevisiae under stressed condition[6]. At present, many methods have been reported for the determination of nucleosides, such as high performance liquid chromatography (HPLC)[7], gas chromatography (GC)[8], capillary electrophoresis (CE)[9] and mass spectrometry (MS)[10]. HPLC and GC could not resolve the co-elution problem of some components, and CE had comparatively inferior detection reproducibility. With the development of chromatography coupled with mass spectrometry (LC-MS), which possess special advantages in complex biological sample analysis because of high separation ability of HPLC for complex matrix compounds and high sensitivity and selectivity of MS. In this study, a sensitive, selective, accurate and fast method was developed for the quantification of 19
Received 21 March 2012; accepted 11 July 2012 * Corresponding author. Email: [email protected] This work was supported by the This work was supported by the National Natural Science Foundation of China (No. 20776029), and the Department of Education of Liaoning Province, China (No. LS2010012). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60622-2
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
nucleosides by rapid resolution liquid chromatography coupled with triple quadrupole tandem mass spectrometry (RRLC-MS/MS). The method could be applied to analyze the changes of the types and contents of nucleosides in E.coli during different growth phases.
2
Experimental
2.1
Apparatuses and materials
1290 Infinity LC system (Agilent, USA) coupled with 6460A Triple Quadrupole mass spectrometry (Agilent, USA) was used for LC-MS system, and the mass spectrometer was equipped with an ESI source. Data acquisition was performed using Masshunter software (Version B.03.01). Electronic balance was Mettler AE 100 (Mettler Toledo, Switzerland). Milli-Q ultrapure water apparatus was purchased from Millipore (Millipore, USA). High speed refrigerated centrifuge was from Kendro (Kendro, Germany). ZHWY- 211C constant temperature incubator and ZHJH-1112 vertical flow clean bench were purchased from Zhicheng, China. MLS-3750 autoclave sterilizer was purchased from SANYO, Japan. Ultrasonic cleaner was from Kunshan Ultrasonic Electronics Co., Ltd. (Kunshan, China). UV-2450 ultraviolet spectrophotometer was purchased from Shimadzu (Shimadzu, Japan). Acetonitrile was of UPLC purity (Merck, Germany). 19 nucleosides standard of mass-grade were purchased from Sigma-Aldrich (Sigma, USA). The following standards were used: pseudouridine, uridine, adenosine, cytosine, 1-methyladenosine, inosine, 2,2-dimethylguanosine, 1-methylguanosine, 1-methylguanosine, N4-acetylcytidine, 6-methyladenosine, guanosine, 5-methyluridine, 5-methylcytidine, 3-methyluridine, 2'-O-methyladenosine, 2'-O-methyluridine, 2'-O-methylcytidine and N6-isopentenyladenosine. RNAiso Plus kits, RNase and diethyl pyrocarbonate (DEPC) were purchased from TaKaRa, Dalian company (TaKaRa, China). 2.2
Strain, culture and culture conditions
Bacterial strain of E.coli AS1.1566 used in this study was provided by 1816 group from Dalian Institute of Chemical Physics. Strains in the analysis were aerobically cultured in nutrient broth (1.0 g L–1 glucose, 10.0 g L–1 peptone, 3.0 g L–1 beef extract and 5.0 g L–1 NaCl). The medium was autoclaved at 121 °C for 15 min before used, and the glucose (through 0.22 μm sterile membrane filter) was added to the culture. The culture was performed in shaking flasks with an agitation rate of 160 rpm at 37 °C. All samples were obtained at different growth phase. 2.3 Liquid chromatographic and mass spectrometric conditions Chromatographic separation was performed with a Waters
ACQUITY UPLC T3 column (100 mm × 2.1 mm × 1.8 μm), the mobile phase consisted of 10 mM ammonium bicarbonate as the mobile phase A and acetonitrile (ACN) as the mobile phase B, at a total flow rate of 0.2 mL min–1. Column temperature was set at 35 °C. The eluent was set to 0% B to 2% B from start to 3 min, after which the proportion of B was changed linearly to 50% during 12 min. From 15 min to 17 min, the proportion of B was raised to 95% and maintained for 3 min to flush the column. After that, the eluent of B was set to the beginning proportion of 0% within 0.1 min for 5 min to equilibrate the system. The injection volume for each sample was 5 µL. The temperature of autosampler was controlled at 4 °C. The mass spectrometer was operated with ESI source and positive mode was chosen to detect nucleosides. The capillary voltage were 4 kV, the flow of the gas from nebulizer was set at 2.76 × 105 Pa. Both of the flow rates of sheath gas and the dry gas from mass to source were set at 8 L min–1, the temperatures of sheath gas and dry gas were controlled at 400 °C and 350 °C. The multiple reaction monitoring (MRM) was used for quantification. 2.4
Total RNA isolation in E.coli
All instruments were soaked overnight with DEPC water and autoclaved for removing RNase pollution in the experiment. A 10-mL E.coli fermentation medium was collected and centrifuged at 9000 g and 4 °C for 5 min in different phase, the supernatant was discarded, then 1 mL RNAiso Plus was added to the centrifuge and vortexed completely. The mixed liquor was transferred into a new tube, holding at room temperature for 5 min. Then centrifuged the sample at 12000 g and 4 °C for 5 min, the supernatant transferred to another tube and mixed with chloroformthe, ratios of these volume was RNAiso Plus: chloroformthe = 5:1, covered the tube and shacked thoroughly for 15 s. Keep the liquor at room temperature for 5 min. The liquor was then recentrifuged at 12000 g and 4 °C for 15 min, and then the supernatant was transferred into a new tube mixing with equal volume of isopropanol. RNA was sedimented by turning the tube upside down thoroughly and keeping at room temperature for 10 min. The pellet was then slowly washed with 1 mL 75% ethanol and centrifuged at 12000 g and 4 °C for 10 min, dried for 2–5 min at room temperature to remove the ethanol. The RNA solutions were obtained by diluting the pellets into 50 μL RNase-free water and stored at –80 °C. 50 U RNase was added to RNA solutions for reaction at 37 °C water bath, keeping for 90 min. Then centrifuged the solutions at 12000 g and 4 °C for 5 min, and then the supernatant was taken for RRLC-MS/MS analysis. 2.5
Date analysis
The nucleosides for E.coli strain were calculated by µg
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
L–1/OD600, and the date analysis was performed according to the protocol of Virginias[11]. 2.6
Preparation of standard solution
The 19 nucleosides standard were prepared respectively for 10 mg in 100 mL RNase-free water, the mixed standard solution was made up to 100 mg L–1 and stored at –20 °C. Calibration curves including different concentration gradients were prepared by diluting standard solution with RNase-free water.
3 3.1
Results and discussion Determination of mass spectrometic conditions
(Fig.1B). Because Waters UPLC HSS T3 bonding utilizes a high strength silica phase at a ligand density that promotes polar compound retention and aqueous mobile phase compatibility (provideing 100% aqueous compatibility), the T3 column was selected for nucleosides separation. Furthermore, when the mobile phase contained 10 mM ammonium acetate (mobile phase A), not only the chromatographic peak of the nucleosides and their separation could be improved, but also their response could be increased. Thus, taking consideration of the polar properties of the necleosides, T3 column was selected with the mobile phase of 10 mM ammonium acetate and acetonitrile (Fig.1C) 3.3
Detection limit and linear range
The 19 mixed standards with the concentration of 10 mg L–1 were analyzed by RRLC-MS/MS system. The total flow rate was 0.2 mL min–1. The mass spectrometry was equipped with electrospray ionization source and scanned in positive mode. Thus, the quasi-molecular ions of 19 nucleosides were detected, also the product ions were selected by optimizing the fragmentor voltage and collision energy. Finally, two ions with best intensity and stability were selected for verification, and the highest one was used for quantification. The MS parameters are shown in Table 1.
The standard solution was prepared as described in Section 2.6. The nucleosides standards were diluted by DEPC water for calibration. The abscissa of calibration curves was the concentration of standards and the ordinate was corresponding peak areas. The linear range, linear regression equation, correlation coefficient and limit of detection (LOD, defined as the signal-to-noise ratio equal to 3) and the limit of quantification (LOQ, defined as the signal-to-noise ratio equal to 10) were obtained by external standard method. The results are summed in Table 2.
3.2
3.4
Selection of chromatographic column
The efficiency of the reversed phase C18 column and HILIC column for the separation of nucleosides was investigated. The results suggested that some nucleosides were eluted in dead-time due to their strong polarity (Fig.1, A and B), and HILIC column had bad response, stability and repeatability
Recovery and precision
The recovery and precision of the method was investigated to verify its applicability. The E.coli samples were harvested in exponential phase, and prepared as described in Section 2.4. However, before processed by RNase, three different concentrations (low, middle and high) of the six nucleosides
Table 1 Conditions of multiple reaction monitoring (MRM) Analyte N6-isopentenyladenosine 2,2-Dimethylguanosine 1-Methylguanosine 1-Methylguanosine N4-acetylcytidine 1-Methyladenosine N6-methyladenosine 2'-O-methyladenosine Inosine Adenosine 3-Methyluridine 5-Methyluridine 2'-O-methyluridine 5-Methylcytidine 2'-O-methylcytidine Pseudouridine Uridine Cytosine Guanosine * For quantification.
Parent ion (m/z)
Product ion (m/z)
Fragmentor (V)
Collisional energy (eV)
336.0 312.1 298.1 298.1 286.1 282.1 282.1 282.1 269.1 268.0 259.1 259.1 259.0 258.1 258.1 245.1 245.1 244.0 152.2
204.2*, 148.1 180.1*, 110.0 166.0*, 149.0 166.0*, 149.0 154.0*, 112.0 150.1*, 94.1 150.1*, 94.1 136.1*, 101.1 137.1*, 177.0 136.1*, 119.0 127.1*, 96.1 127.1*, 96.1 113.1*, 147.1 126.1*, 109.0 112.1*, 195.0 125.0*, 191.0 113.1*, 70.1 112.0*, 203.0 135.1*, 110.1
100 100 70 80 80 90 90 100 70 70 70 70 70 80 80 70 70 80 80
15 8 10 10 5 20 20 10 10 20 5 5 5 10 5 10 5 5 10
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
Fig.1 Extracted ion chromatogram of 19 nucleoside standards by RRLC-MS/MS in MRM mode with three kinds of columns A: C18, B: HILIC, C:T3,1: pseudouridine, 2: Cytosine, 3: Uridine, 4: 1-methyladenosine, 5: 5-methylcytidine, 6: 7-methylguanosine, 7: Guanosine, 8; 2'-O-methylcytidine, 9: Inosine, 10: N4-acetylcytidine, 11: 5-methyluridine, 12: 2'-O-methyluridine, 13: 1-methylguanosine, 14: 3-methyluridine, 15: Adenosine, 16: 2,2-dimethylguanosine, 17: 2'-O-methyladenosine, 18: 6-methyladenosine, 19: N6-isopentenyladenosine
Table 2 Linear regression equations, correlation coefficients (R2), linear ranges, LOQs and LODs of 19 nucleosides Linear regression equation
Linear range (µg L–1)
N6-isopentenyladenosine
Y=403.320X+56.406
2,2-Dimethylguanosine
Y=17.646X+131.990
1-Methylguanosine
Y=14.875X+115.540
1-Methylguanosine
Analyte
Correlation coefficient (R2)
LOQ (µg L–1)
LOD (µg L–1)
0.1–200
0.9983
0.05
0.02
1–500
0.9975
0.5
0.1
1–500
0.9946
0.5
0.1
Y=12.537X+135.310
1–1000
0.9974
0.5
0.1
N4-acetylcytidine
Y=0.107X+3,451
10–2000
0.9990
10
5
1-Methyladenosine
Y=84.272X+494.460
1–500
0.9972
0.5
0.1
6-Methyladenosine
Y=36.591X+54.760
0.1–100
0.9994
0.1
0.05
2'-O-methyladenosine
Y=69.799X+254.370
1–200
0.9962
0.5
0.1
Y=1.414X+31.395
0.5–500
0.9976
0.4
0.1
Adenosine
Y=21.787X+226.830
1–200
0.9969
0.2
0.1
3-Methyluridine
Y=1.154X+150.360
10–1000
0.9953
5
1
5-Methyluridine
Y=1.074X+94.447
10–5000
0.9950
5
2
2'-O-methyluridine
Y=2.325X+47.490
10–2000
0.9989
5
2
5-Methylcytidine
Y=22.034X+26.861
2–200
0.9997
1
0.1
2'-O-methylcytidine
Y=9.285X+70.353
1–500
0.9989
0.4
0.1
Y=1.413X-7.223
0.1–100
0.9999
0.1
0.05
Uridine
Y=0.426X+12.317
20–5000
0.9995
20
10
Cytosine
Y=4.563X+54.913
0.1–200
0.9992
0.1
0.05
Guanosine
Y=0.036X+18.877
1–1000
0.9916
0.5
0.1
Inosine
Pseudouridine
standards were added to the prepared samples. Every sample was prepared for four biological replicates. The recovery and intra-day precision (RSD) are displayed in Table 3. The ranges of recovery and relative standard deviation (RSD) were in the range of 79.0%–119.8% and 2.8%–14.0%, respectively.
3.5
Nucleosides detection in E.coli
The levels of nucleosides in E.coli from different phases were analyzed by the LC/MS method in this study. The samples in exponential phase, the time between exponential
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
and stationary phase, stationary phase and decline phase were selected, which were after inoculation 4, 8, 12 and 24 h, respectively. Seven nucleosides containing cytosine, 5-methylcytidine, guanosine, adenosine, inosine, 6-methyladenosine and uridine could be detected at different phases (Fig.2A). The amount of guanosine, inosine and
adenosine were much higher than that of the others. Additionally, the distinct change of guanosine during the growth phase was observed by using significant analysis of micromatrix (SAM) analysis (Fig.2B, Δ = 0.05, p < 0.05). As shown in Fig.2C, the level of guanosine was significantly decreased with the growth of E.coli.
Table 3 Recoveries and precision of nucleosides in E.coli Analyte
Control sample (µg L–1)
6-Methyladenosine
0.37
Inosine
7.59
Adenosine
9.06
Uridine
0.70
Cytosine
0.18
Guanosine
17.98
Added (µg L–1)
Found (µg L–1)
Recovery (%, n = 4)
RSD (%, n = 4)
0.16 0.40 0.80 3.43 8.57 17.14 4.29 10.71 21.43 0.30 0.76 1.51 0.086 0.21 0.43 3.86 9.64 19.29
0.53 0.74 1.24 11.09 17.42 25.22 14.12 20.91 33.19 0.97 1.61 2.33 0.25 0.42 0.64 21.56 29.16 34.31
100.2 93.0 110.9 100.5 114.7 102.8 116.6 110.6 102.8 93.2 119.8 107.8 79.0 114.1 108.8 98.4 116.1 84.7
7.6 10.9 10.6 7.2 4.7 9.2 9.4 14.0 9.2 2.8 5.1 3.5 7.5 8.7 6.9 9.7 4.8 8.5
Fig.2 Quantification of intercellular nucleosides: (A) Concentration of 7 detectable nucleosides in E.coli at different phases; (B) Significant analysis of the quantitated nucleosides and (C) Time course change of intercellular guanosine
Researchers have confirmed that various physiological functions and the growth statuses of E. coli have relation with the relative content of nucleotides. Most of studies were focused on the regulation of guanosine pyrophosphate (ppGpp) to metabolism. However, little information about nucleosides
and the growth status in E.coli was investigated. In this study, we discovered that the significant change of guanosine occurred during the growth of E.coli. It is similar with the reports of Buckstein, which discovered the total amount of nucleotides reduced when the E.coli in logarithmic phase
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
entered into stationary phase[12]. The research also found that the variation trend of guanosine was probably caused by the consumption during the production of guanosine pyrophosphates[13]. The continuous decrease of guanosine in E.coli from logarithmic phase to stationary phase was contributed to the growth inhibitory effect (Fig.2C), which was agree with the results that some researchers obtained. In addition, guanosine could be used as one of carbon sources during the growth of E.coli. Thus, the decrease of guanosine may be caused by the consumption of carbon source[14]. Generally, the further research about the mechanism of the change of guanosine and regulation to the growth of E.coli need more detailed insights in the future.
K, Janusz M B, Henri G, Kristian R. Nuleic. Acids Res., 2009, 37: D118–D121 [2]
2006, 5(1): 87–95 [3]
Connie Y, Hannah T, Wojciech C, Elzbieta S, Andrzej J M, Richard G, Agnieszka M, Paul F A. J. Biol. Chem., 2002, 277(19): 16391–16395
[5]
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Conclusions
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[8]
In this study, a new method with rapid separation and simplicity was used for the determination of 19 nucleosides. The method had better sensitivity, repeatability and accuracy for the quantification of nucleosides. In this study, seven nucleosides could be readily quantified in E. coli at different phases. Additionally, distinct decrease of the guanosine during the growth phase was discovered. The method could be used for the analysis of nucleosides in microorganism.
Agris P F, Vendeix F P, Graham W D. J. Mol. Biol., 2007, 366: 1–13
[4]
[7]
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Meng Z J, Patrick A L. Brief. Funct. Genomic. Proteomic.,
James I L,Tom D M, Salah E, Andrew F, Karl H S, Russell P N. Rapid Commun. Mass Sp., 1993, 7: 427–434
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Wang S F, Zhao X P, Mao Y, Cheng Y Y. J. Chromatogr. A., 2007, 1147(2): 254–260
[10]
Elaine M T, Donald J L J, John H. L, Debbie A M, Peter B F,
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Virginia G T, Robert T, Gilbert C. Natl. Acad. Sci. USA., 2001,
Karen B. Rapid Commun Mass Sp., 2008, 22: 19–28 98(9): 5116–5121 [12]
Buckstein M H, He J, Rubin H. J. Bacteriol., 2008, 190: 718–726
References [1]
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[13]
Petersen C. J. Biol. Chem., 1999, 274(9): 5348–5356
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Schuch R, Garibian A, Nygaard P, Raymond S, Araik G, Hans H S, Patrick J P, Per N. Microbiology, 1999, 145: 2957–2966
Cite this article as: Chin J Anal Chem, 2013, 41(1), 36–41.
RESEARCH PAPER
Determination of Nucleosides in Escherichia coli by Rapid Resolution Liquid Chromatography-Tandem Quadrupole Mass Spectrometry XIE Yu-Ping1,2, TIAN Jing1,*, GAO Peng2, XU GUO-Wang2, FEI Xu1, WANG Yi1 1 2
School of Bioengineering, Dalian Polytechnic University, Dalian 116034, China Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Abstract: A sensitive and selective method was developed for the quantitative determination of nucleosides in Escherichia coli. An Agilent 1290 HPLC was used with mobile phase A (10 mM ammonium acetate) and mobile phase B (acetonitrile) at a flow-rate of 0.2 mL min–1 coupled with a mass spectrometry scanned in multiple reaction monitoring (MRM) mode. In the result, the limit of quantification and detection of 19 nucleosides were in the range of 0.05–20 µg L–1 and 0.02–10 µg L–1. The linearity of the detected nucleosides was excellent with R2 > 0.99 and the limits of detection were all satisfied. The recoveries for the compounds were between 79.0%–119.8%, and the relative standard deviation was below 14%. The method was capable of quantitation 7 nucleosides in Escherichia coli, of which guanosine was found to decrease along with culture phases. Key Words: Rapid resolution liquid chromatography-tandem mass spectrometry; Nucleoside; Escherichia Coli
1
Introduction
Nucleic acids that exist in living cells include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and the latter mainly exists in the form of ribosomal RNA, messenger RNA and transfer RNA(tRNA). Besides four normal bases, there are also some minor bases in tRNA, which are usually generated by chemical modification of the four major ones during post-transcription or in exceptional cases. Decades of studies have revealed mainly about 100 ribonucleoside structures incorporated as post-transcriptional modifications across all organisms and more than 40 in the prokaryotes[1,2]. The effect of modified nucleosides on RNA function is still unknown. In general, tRNA modifications enhance ribosome binding affinity, reduce misreading and modulate frame-shifting, all of which affect the rate and
fidelity of translation[3–5]. Recent studies suggested that modified nucleosides could improve the survival ability of Saccharomyces cerevisiae under stressed condition[6]. At present, many methods have been reported for the determination of nucleosides, such as high performance liquid chromatography (HPLC)[7], gas chromatography (GC)[8], capillary electrophoresis (CE)[9] and mass spectrometry (MS)[10]. HPLC and GC could not resolve the co-elution problem of some components, and CE had comparatively inferior detection reproducibility. With the development of chromatography coupled with mass spectrometry (LC-MS), which possess special advantages in complex biological sample analysis because of high separation ability of HPLC for complex matrix compounds and high sensitivity and selectivity of MS. In this study, a sensitive, selective, accurate and fast method was developed for the quantification of 19
Received 21 March 2012; accepted 11 July 2012 * Corresponding author. Email: [email protected] This work was supported by the This work was supported by the National Natural Science Foundation of China (No. 20776029), and the Department of Education of Liaoning Province, China (No. LS2010012). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60622-2
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
nucleosides by rapid resolution liquid chromatography coupled with triple quadrupole tandem mass spectrometry (RRLC-MS/MS). The method could be applied to analyze the changes of the types and contents of nucleosides in E.coli during different growth phases.
2
Experimental
2.1
Apparatuses and materials
1290 Infinity LC system (Agilent, USA) coupled with 6460A Triple Quadrupole mass spectrometry (Agilent, USA) was used for LC-MS system, and the mass spectrometer was equipped with an ESI source. Data acquisition was performed using Masshunter software (Version B.03.01). Electronic balance was Mettler AE 100 (Mettler Toledo, Switzerland). Milli-Q ultrapure water apparatus was purchased from Millipore (Millipore, USA). High speed refrigerated centrifuge was from Kendro (Kendro, Germany). ZHWY- 211C constant temperature incubator and ZHJH-1112 vertical flow clean bench were purchased from Zhicheng, China. MLS-3750 autoclave sterilizer was purchased from SANYO, Japan. Ultrasonic cleaner was from Kunshan Ultrasonic Electronics Co., Ltd. (Kunshan, China). UV-2450 ultraviolet spectrophotometer was purchased from Shimadzu (Shimadzu, Japan). Acetonitrile was of UPLC purity (Merck, Germany). 19 nucleosides standard of mass-grade were purchased from Sigma-Aldrich (Sigma, USA). The following standards were used: pseudouridine, uridine, adenosine, cytosine, 1-methyladenosine, inosine, 2,2-dimethylguanosine, 1-methylguanosine, 1-methylguanosine, N4-acetylcytidine, 6-methyladenosine, guanosine, 5-methyluridine, 5-methylcytidine, 3-methyluridine, 2'-O-methyladenosine, 2'-O-methyluridine, 2'-O-methylcytidine and N6-isopentenyladenosine. RNAiso Plus kits, RNase and diethyl pyrocarbonate (DEPC) were purchased from TaKaRa, Dalian company (TaKaRa, China). 2.2
Strain, culture and culture conditions
Bacterial strain of E.coli AS1.1566 used in this study was provided by 1816 group from Dalian Institute of Chemical Physics. Strains in the analysis were aerobically cultured in nutrient broth (1.0 g L–1 glucose, 10.0 g L–1 peptone, 3.0 g L–1 beef extract and 5.0 g L–1 NaCl). The medium was autoclaved at 121 °C for 15 min before used, and the glucose (through 0.22 μm sterile membrane filter) was added to the culture. The culture was performed in shaking flasks with an agitation rate of 160 rpm at 37 °C. All samples were obtained at different growth phase. 2.3 Liquid chromatographic and mass spectrometric conditions Chromatographic separation was performed with a Waters
ACQUITY UPLC T3 column (100 mm × 2.1 mm × 1.8 μm), the mobile phase consisted of 10 mM ammonium bicarbonate as the mobile phase A and acetonitrile (ACN) as the mobile phase B, at a total flow rate of 0.2 mL min–1. Column temperature was set at 35 °C. The eluent was set to 0% B to 2% B from start to 3 min, after which the proportion of B was changed linearly to 50% during 12 min. From 15 min to 17 min, the proportion of B was raised to 95% and maintained for 3 min to flush the column. After that, the eluent of B was set to the beginning proportion of 0% within 0.1 min for 5 min to equilibrate the system. The injection volume for each sample was 5 µL. The temperature of autosampler was controlled at 4 °C. The mass spectrometer was operated with ESI source and positive mode was chosen to detect nucleosides. The capillary voltage were 4 kV, the flow of the gas from nebulizer was set at 2.76 × 105 Pa. Both of the flow rates of sheath gas and the dry gas from mass to source were set at 8 L min–1, the temperatures of sheath gas and dry gas were controlled at 400 °C and 350 °C. The multiple reaction monitoring (MRM) was used for quantification. 2.4
Total RNA isolation in E.coli
All instruments were soaked overnight with DEPC water and autoclaved for removing RNase pollution in the experiment. A 10-mL E.coli fermentation medium was collected and centrifuged at 9000 g and 4 °C for 5 min in different phase, the supernatant was discarded, then 1 mL RNAiso Plus was added to the centrifuge and vortexed completely. The mixed liquor was transferred into a new tube, holding at room temperature for 5 min. Then centrifuged the sample at 12000 g and 4 °C for 5 min, the supernatant transferred to another tube and mixed with chloroformthe, ratios of these volume was RNAiso Plus: chloroformthe = 5:1, covered the tube and shacked thoroughly for 15 s. Keep the liquor at room temperature for 5 min. The liquor was then recentrifuged at 12000 g and 4 °C for 15 min, and then the supernatant was transferred into a new tube mixing with equal volume of isopropanol. RNA was sedimented by turning the tube upside down thoroughly and keeping at room temperature for 10 min. The pellet was then slowly washed with 1 mL 75% ethanol and centrifuged at 12000 g and 4 °C for 10 min, dried for 2–5 min at room temperature to remove the ethanol. The RNA solutions were obtained by diluting the pellets into 50 μL RNase-free water and stored at –80 °C. 50 U RNase was added to RNA solutions for reaction at 37 °C water bath, keeping for 90 min. Then centrifuged the solutions at 12000 g and 4 °C for 5 min, and then the supernatant was taken for RRLC-MS/MS analysis. 2.5
Date analysis
The nucleosides for E.coli strain were calculated by µg
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
L–1/OD600, and the date analysis was performed according to the protocol of Virginias[11]. 2.6
Preparation of standard solution
The 19 nucleosides standard were prepared respectively for 10 mg in 100 mL RNase-free water, the mixed standard solution was made up to 100 mg L–1 and stored at –20 °C. Calibration curves including different concentration gradients were prepared by diluting standard solution with RNase-free water.
3 3.1
Results and discussion Determination of mass spectrometic conditions
(Fig.1B). Because Waters UPLC HSS T3 bonding utilizes a high strength silica phase at a ligand density that promotes polar compound retention and aqueous mobile phase compatibility (provideing 100% aqueous compatibility), the T3 column was selected for nucleosides separation. Furthermore, when the mobile phase contained 10 mM ammonium acetate (mobile phase A), not only the chromatographic peak of the nucleosides and their separation could be improved, but also their response could be increased. Thus, taking consideration of the polar properties of the necleosides, T3 column was selected with the mobile phase of 10 mM ammonium acetate and acetonitrile (Fig.1C) 3.3
Detection limit and linear range
The 19 mixed standards with the concentration of 10 mg L–1 were analyzed by RRLC-MS/MS system. The total flow rate was 0.2 mL min–1. The mass spectrometry was equipped with electrospray ionization source and scanned in positive mode. Thus, the quasi-molecular ions of 19 nucleosides were detected, also the product ions were selected by optimizing the fragmentor voltage and collision energy. Finally, two ions with best intensity and stability were selected for verification, and the highest one was used for quantification. The MS parameters are shown in Table 1.
The standard solution was prepared as described in Section 2.6. The nucleosides standards were diluted by DEPC water for calibration. The abscissa of calibration curves was the concentration of standards and the ordinate was corresponding peak areas. The linear range, linear regression equation, correlation coefficient and limit of detection (LOD, defined as the signal-to-noise ratio equal to 3) and the limit of quantification (LOQ, defined as the signal-to-noise ratio equal to 10) were obtained by external standard method. The results are summed in Table 2.
3.2
3.4
Selection of chromatographic column
The efficiency of the reversed phase C18 column and HILIC column for the separation of nucleosides was investigated. The results suggested that some nucleosides were eluted in dead-time due to their strong polarity (Fig.1, A and B), and HILIC column had bad response, stability and repeatability
Recovery and precision
The recovery and precision of the method was investigated to verify its applicability. The E.coli samples were harvested in exponential phase, and prepared as described in Section 2.4. However, before processed by RNase, three different concentrations (low, middle and high) of the six nucleosides
Table 1 Conditions of multiple reaction monitoring (MRM) Analyte N6-isopentenyladenosine 2,2-Dimethylguanosine 1-Methylguanosine 1-Methylguanosine N4-acetylcytidine 1-Methyladenosine N6-methyladenosine 2'-O-methyladenosine Inosine Adenosine 3-Methyluridine 5-Methyluridine 2'-O-methyluridine 5-Methylcytidine 2'-O-methylcytidine Pseudouridine Uridine Cytosine Guanosine * For quantification.
Parent ion (m/z)
Product ion (m/z)
Fragmentor (V)
Collisional energy (eV)
336.0 312.1 298.1 298.1 286.1 282.1 282.1 282.1 269.1 268.0 259.1 259.1 259.0 258.1 258.1 245.1 245.1 244.0 152.2
204.2*, 148.1 180.1*, 110.0 166.0*, 149.0 166.0*, 149.0 154.0*, 112.0 150.1*, 94.1 150.1*, 94.1 136.1*, 101.1 137.1*, 177.0 136.1*, 119.0 127.1*, 96.1 127.1*, 96.1 113.1*, 147.1 126.1*, 109.0 112.1*, 195.0 125.0*, 191.0 113.1*, 70.1 112.0*, 203.0 135.1*, 110.1
100 100 70 80 80 90 90 100 70 70 70 70 70 80 80 70 70 80 80
15 8 10 10 5 20 20 10 10 20 5 5 5 10 5 10 5 5 10
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Fig.1 Extracted ion chromatogram of 19 nucleoside standards by RRLC-MS/MS in MRM mode with three kinds of columns A: C18, B: HILIC, C:T3,1: pseudouridine, 2: Cytosine, 3: Uridine, 4: 1-methyladenosine, 5: 5-methylcytidine, 6: 7-methylguanosine, 7: Guanosine, 8; 2'-O-methylcytidine, 9: Inosine, 10: N4-acetylcytidine, 11: 5-methyluridine, 12: 2'-O-methyluridine, 13: 1-methylguanosine, 14: 3-methyluridine, 15: Adenosine, 16: 2,2-dimethylguanosine, 17: 2'-O-methyladenosine, 18: 6-methyladenosine, 19: N6-isopentenyladenosine
Table 2 Linear regression equations, correlation coefficients (R2), linear ranges, LOQs and LODs of 19 nucleosides Linear regression equation
Linear range (µg L–1)
N6-isopentenyladenosine
Y=403.320X+56.406
2,2-Dimethylguanosine
Y=17.646X+131.990
1-Methylguanosine
Y=14.875X+115.540
1-Methylguanosine
Analyte
Correlation coefficient (R2)
LOQ (µg L–1)
LOD (µg L–1)
0.1–200
0.9983
0.05
0.02
1–500
0.9975
0.5
0.1
1–500
0.9946
0.5
0.1
Y=12.537X+135.310
1–1000
0.9974
0.5
0.1
N4-acetylcytidine
Y=0.107X+3,451
10–2000
0.9990
10
5
1-Methyladenosine
Y=84.272X+494.460
1–500
0.9972
0.5
0.1
6-Methyladenosine
Y=36.591X+54.760
0.1–100
0.9994
0.1
0.05
2'-O-methyladenosine
Y=69.799X+254.370
1–200
0.9962
0.5
0.1
Y=1.414X+31.395
0.5–500
0.9976
0.4
0.1
Adenosine
Y=21.787X+226.830
1–200
0.9969
0.2
0.1
3-Methyluridine
Y=1.154X+150.360
10–1000
0.9953
5
1
5-Methyluridine
Y=1.074X+94.447
10–5000
0.9950
5
2
2'-O-methyluridine
Y=2.325X+47.490
10–2000
0.9989
5
2
5-Methylcytidine
Y=22.034X+26.861
2–200
0.9997
1
0.1
2'-O-methylcytidine
Y=9.285X+70.353
1–500
0.9989
0.4
0.1
Y=1.413X-7.223
0.1–100
0.9999
0.1
0.05
Uridine
Y=0.426X+12.317
20–5000
0.9995
20
10
Cytosine
Y=4.563X+54.913
0.1–200
0.9992
0.1
0.05
Guanosine
Y=0.036X+18.877
1–1000
0.9916
0.5
0.1
Inosine
Pseudouridine
standards were added to the prepared samples. Every sample was prepared for four biological replicates. The recovery and intra-day precision (RSD) are displayed in Table 3. The ranges of recovery and relative standard deviation (RSD) were in the range of 79.0%–119.8% and 2.8%–14.0%, respectively.
3.5
Nucleosides detection in E.coli
The levels of nucleosides in E.coli from different phases were analyzed by the LC/MS method in this study. The samples in exponential phase, the time between exponential
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
and stationary phase, stationary phase and decline phase were selected, which were after inoculation 4, 8, 12 and 24 h, respectively. Seven nucleosides containing cytosine, 5-methylcytidine, guanosine, adenosine, inosine, 6-methyladenosine and uridine could be detected at different phases (Fig.2A). The amount of guanosine, inosine and
adenosine were much higher than that of the others. Additionally, the distinct change of guanosine during the growth phase was observed by using significant analysis of micromatrix (SAM) analysis (Fig.2B, Δ = 0.05, p < 0.05). As shown in Fig.2C, the level of guanosine was significantly decreased with the growth of E.coli.
Table 3 Recoveries and precision of nucleosides in E.coli Analyte
Control sample (µg L–1)
6-Methyladenosine
0.37
Inosine
7.59
Adenosine
9.06
Uridine
0.70
Cytosine
0.18
Guanosine
17.98
Added (µg L–1)
Found (µg L–1)
Recovery (%, n = 4)
RSD (%, n = 4)
0.16 0.40 0.80 3.43 8.57 17.14 4.29 10.71 21.43 0.30 0.76 1.51 0.086 0.21 0.43 3.86 9.64 19.29
0.53 0.74 1.24 11.09 17.42 25.22 14.12 20.91 33.19 0.97 1.61 2.33 0.25 0.42 0.64 21.56 29.16 34.31
100.2 93.0 110.9 100.5 114.7 102.8 116.6 110.6 102.8 93.2 119.8 107.8 79.0 114.1 108.8 98.4 116.1 84.7
7.6 10.9 10.6 7.2 4.7 9.2 9.4 14.0 9.2 2.8 5.1 3.5 7.5 8.7 6.9 9.7 4.8 8.5
Fig.2 Quantification of intercellular nucleosides: (A) Concentration of 7 detectable nucleosides in E.coli at different phases; (B) Significant analysis of the quantitated nucleosides and (C) Time course change of intercellular guanosine
Researchers have confirmed that various physiological functions and the growth statuses of E. coli have relation with the relative content of nucleotides. Most of studies were focused on the regulation of guanosine pyrophosphate (ppGpp) to metabolism. However, little information about nucleosides
and the growth status in E.coli was investigated. In this study, we discovered that the significant change of guanosine occurred during the growth of E.coli. It is similar with the reports of Buckstein, which discovered the total amount of nucleotides reduced when the E.coli in logarithmic phase
XIE Yu-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(1): 36–41
entered into stationary phase[12]. The research also found that the variation trend of guanosine was probably caused by the consumption during the production of guanosine pyrophosphates[13]. The continuous decrease of guanosine in E.coli from logarithmic phase to stationary phase was contributed to the growth inhibitory effect (Fig.2C), which was agree with the results that some researchers obtained. In addition, guanosine could be used as one of carbon sources during the growth of E.coli. Thus, the decrease of guanosine may be caused by the consumption of carbon source[14]. Generally, the further research about the mechanism of the change of guanosine and regulation to the growth of E.coli need more detailed insights in the future.
K, Janusz M B, Henri G, Kristian R. Nuleic. Acids Res., 2009, 37: D118–D121 [2]
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Connie Y, Hannah T, Wojciech C, Elzbieta S, Andrzej J M, Richard G, Agnieszka M, Paul F A. J. Biol. Chem., 2002, 277(19): 16391–16395
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[8]
In this study, a new method with rapid separation and simplicity was used for the determination of 19 nucleosides. The method had better sensitivity, repeatability and accuracy for the quantification of nucleosides. In this study, seven nucleosides could be readily quantified in E. coli at different phases. Additionally, distinct decrease of the guanosine during the growth phase was discovered. The method could be used for the analysis of nucleosides in microorganism.
Agris P F, Vendeix F P, Graham W D. J. Mol. Biol., 2007, 366: 1–13
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