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Comparison of Two Detection Methods for Fragment Ions of Escherichia coli Carbon Center Metabolites.


In studies of metabolic processes in microorganisms, it's very important to detect target metabolite, both qualitatively and quantitatively. Detecting target metabolite precisely and determining its content are important goals in modern metabolomics. Such studies have progressed significantly in recent years as a result of new and improved methodologies (1-4).

The aim of this study is to analyse Escherichia coli carbon center metabolites by High Performance Liquid Chromatography, coupled with Tandem Mass Spectrometry. Escherichia coli is commonly used for experimental purposes, and particularly in genetic engineering. There have been 904 different Escherichia coli metabolites mentioned in research papers, involving in 932 different reactions (5-6), the number of which seems to be rapidly increasing as more research follows. Carbon central metabolism are common to many reactions in Escherichia coli and are therefore important to study to further understand the metabolomics of this commonly used bacterium. This study is to establish the experimental conditions of High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry for Escherichia coli carbon center metabolites. In addition, we hope to provide some references with regards to cell extraction methods and detection, for future studies.

There have been many studies describing extraction methods of mesostate in TCA and modern analysis techniques for phosphorylation products in most of the central metabolic pathways. Gas chromatography coupled with mass spectrometry is very common in metabolite detection, but its disadvantage is that heat-sensitive compounds have denatured decomposition more easily during the detection. Both triple quadrupole mass spectrometry and ion trap mass spectrometry are multi-stage mass spectrometry. Triple quadrupole mass spectrometry is mainly used for quantitative and qualitative analysis of target compounds and is often referred to as space tandem mass spectrometry. Ion trap mass spectrometry is time tandem mass spectrometry which is generally used for qualitative analysis and structural analysis of unknown compounds (7-9).

Recently, with the development of MS and interface technology of LC/MS, it is now possible to determine conjugated metabolites directly. It has been reported that glucuronide conjugates can be determined directly by LC/MS/ MS. The basis for the structure of this conjugate is that the new fragment ions are found when a molecular ion peak draws off 176u in the secondary mass (10). In this study, we set out to explore and establish analysis of Escherichia coli carbon center metabolites using high performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). It is one of the most important methods for standard sample detection of these kinds of metabolites.


Main Instruments

Triple quadrupole mass spectrometer; ion trap mass spectrometer; sonicator; electronic analytical balance

Experimental Methods

Chromatographic conditions

Chromatographic column: 250mm x 4.6mm x 5um; column temperature: 35[degrees]C capillary temperature: 380[degrees]C; molecular weight range: 50-400[degrees]C mobile phase A: 10mmol/L ammonium acetate solution; mobile phase B: acetonitrile

Mass spectrometry conditions

negative ion scan; electrospray ionization voltage (ESI): 4.5 kV ion source temperature: 480[degrees]C; injection volume: 5 ml

Mass spectrometry conditions

Negative ion scan; electrospray ionization voltage (ESI): 4.5 kVion source temperature: 480!; injection volume: 5 ml

Preparation standard solution of samples

We prepared ten kinds of metabolites, which are pyruvic acid; glucose-6-phosphate dipotassium salt; fructose-6-phosphate dipotassium salt; succinic acid; aspartic acid; isocitric acid trisodium salt; citric acid; glutamate acid; [alpha]-ketoglutarate acid; fumaric acid acid respectively. To make all the standard solutions, use an electronic analytical balance to enable highly accurate weighing.


Results of ion trap LC

X-axis represents ion response; Y-axis represents the time respectively; ESI: electrospray ionization voltage; MRM-multistage reaction detection; Frag-collision voltage; CID-collisional dissociation voltage

Compared with Fig. 5 and Fig. 6, Fig. 1 and Fig. 2 show that only under a collision-induced voltage of -20 eV can fluid glutamate --prospective parent ion 146.1, be broken down for Sub ion and detected by an ion trap mass spectrometer. However, glutamate-prospective parent ion 146 detected by liquid chromatography triple quadrupole mass spectrometer could be broken down for Sub ion if the collision-induced voltage was -15 eV. So, the required collision-induced voltage for a Gas chromatography-ion trap mass spectrometry instrument is lower. The chromatomap illustrates the differences in the peak time. Fig. 1 (MS/MS) shows that glutamic acid appears at 4.18 min, whereas in Fig. 5 (GC-ITMS) the time to appearance is 3.76 min. This suggests that the Gas chromatography-ion trap mass spectrometry instrument can make the glutamic acid peak time earlier. From Fig. 2 it can be seen that the sub ion is 128.1 and in Fig. 6 it is 84.0 and 102.1. This suggests that MS/MS and GC-ITMS can both be used in multistage mass spectrometry, and that GC-ITMS can obtain smaller sub ions at a lower collision energy.

Comparing Fig. 1 and Fig. 3 with Fig. 7 and Fig. 8, we can learn that the collision energy of breaking citrate prospective parent ion 191 in MS/MS is -25 eV and in GC-ITMS is only -15eV. Comparing the chromatograms in Fig. 1 and Fig. 7, the peak of citrate in Fig. 7 is sharper and fuller than it is in Fig. 1. Sub ion in Fig. 3 is 87.3 and in Fig. 8 is 111.0. So, the two mass spectrometry methods can both be used in a multi-stage mass spectrometry, but in contrast, GC-ITMS can get an improved peak for Citric acid detection.

Comparing Fig. 1 and Fig. 4 with Fig. 9 and Fig. 10, collision energy of breaking pyruvate parent ion 87.0 is -10eV in MS/MS and is only -7eV in GC-ITMS. The pyruvate peak in Fig. 9 is sharper and more beautiful than it is in Fig. 1. and there is no broken parent ion 87.1 in Fig. 4, but in Fig. 10 sub ion we can see that 43.0 appears in the spectrum. The minimum mass set by the ion trap LC-MS instrument is 50, which means that any fragment ions below 50 cannot be detected. It is possible that the collision energy produced by Ion Trap is too small to detect pyruvate fragment ions by GC-ITMS. In that case, GC-ITMS would be a better way to detect pyruvate.


In conclusion, using GC-ITMS we can obtain smaller particle size fragment ions. Relatively speaking, detecting metabolite fragment ions by GC-ITMS is an improved method to obtain a clearer peak. 10.22207/JPAM.12.4.05


This work was supported by the Chinese National Natural Science Foundation [grants 31200626 and 31460233, 31372402); the Biology Key Subject Construction of Anhui (2014SKQJ017).


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Bin Rui [1] *, Xiaotao Wei [2], Long Chen [1], Yun An [3] and Han Wen [1]

[1] School of Life Science, Anhui Agricultural University, 230026, Hefei, China. [2] School of Information and Computer, Anhui Agricultural University, 230026, Hefei, China. [3] School of Tea and Food Technology, Anhui Agricultural University, 230026, Hefei, China.

* Correspondence:

(Received: 08 July 2018; accepted: 12 September 2018)

Caption: Fig. 1. Mixing spectrum of ten different metabolites.

Caption: Fig. 2. Spectrum of glutamate acid with ion trap LC

Caption: Fig. 3. Spectrum of citric acid with ion trap LC

Caption: Fig. 4. Spectrum of pyruvic acid with ion trap LC

Caption: Fig. 5. Chromatogram of glutamate acid with triple quadrupole LC

Caption: Fig. 6. Spectrum of glutamate acid with triple quadrupole LC

Caption: Fig. 7. Chromatogram of citric acid with triple quadrupole LC

Caption: Fig. 8. Spectrum. of citric acid with triple quadrupole LC

Caption: Fig. 9. Chromatogram of pyruvic acid with triple quadrupole LC

Caption: Fig. 10. Spectrum. of pyruvic acid with triple quadrupole LC
Table 1. Gradient elution parameters

Time   Mobile phase A   Mobile phase B

0            0               100
3            10               90
15           50               50
25           50               50
27           90               10
35          100               0

Table 2. The parameters of ten kinds of metabolites detected by ion
trap mass spectrometry

Metabolites                               parent      ion       CE

Citric acid                                191        87       25eV
D-Frutose-6-phosphate dipotassium salt     259        97       27eV
Dipotassium D-Glucose-6-phosphate          259      97078.9    27eV
2-ketoglutaric acid                        145        101      18eV
Glutamic acid                              146        128      20eV
Fumaric acid                               115        71       20eV
Pyruvic acid                                87        87       10eV
Aspartic acid                              132        88       27eV
Succitric acid                             117        73       27eV
Isocitric acid trisodium salt hydrate      191     1110173.1   27eV

Table 3. The parameters of ten kinds of metabolites detected by triple
quadrupole mass spectrometry

Metabolites                          CE     parent       ion (m/z)
                                           ion (m/z)

Dipotassium D-Glucose-6-phosphate    135      259      139, 169, 199
Aspartic acid                        135      132       71.2, 88.1
Citric acid                          135      191          111.0
D-Frutose-6-phosphate dipotassium    135      259        139, 169
Pyruvic acid                         135      87           43.0
Succitric acid                       135      117          73.1
Isocitric acid trisodium salt        135      191       73.1, 167.1
Glutamic acid                        135      146       84.0, 102.1
2-ketoglutaric acid                  135      145       57.1, 145.1
Fumaric acid                         135      115          71.0
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Article Details
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Author:Rui, Bin; Wei, Xiaotao; Chen, Long; An, Yun; Wen, Han
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Dec 1, 2018
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