Optimized sampling protocol for mass spectrometry-based metabolomics in Streptomyces
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KeywordsQuantitative metabolomics Streptomyces Quenching Leakage Extraction
molar transition energy
gas chromatography–mass spectrometry
isotope dilution mass spectrometry
liquid chromatography–mass spectrometry
intracellular metabolites extracted from cell pellets
acetone:base solution (acetone:ethanol = 1:1, mol/mol) = 5:1 (v/v)
- IsoaB, isoamylol
base solution (acetone:ethanol = 1:1, mol/mol) = 5:1 (v/v)
- ProB, propanol
base solution (acetone:ethanol = 1:1, mol/mol) = 5:1 (v/v)
- MethB methanol
base solution (acetone:ethanol = 1:1, mol/mol) = 5:1 (v/v)
selected ion monitoring
dry cell weight
ultra-high-performance liquid chromatography–mass, tandem–mass spectrometry
selected reaction monitoring
Streptomyces, which belong to prokaryotes but show filamentous growth, are the most noteworthy producers of novel medicines for resisting various diseases including cancers, immunological diseases, and infections caused by bacteria, fungi, viruses and parasites. In the 1950s and 60 s, following the golden era of antibiotics discovery, around 70 to 80% of antibiotics that treated bacteria and fungi were isolated from diverse Streptomyces (Berdy 2005). At present, Streptomyces continue to attract attention due to the need to discover new medicines to break the drug resistance of pathogenic micro-organism (Hwang et al. 2014). As a result of this significant history, plenty of researchers have focused on Streptomyces. However, many of their mechanisms still need to be explained. As a result, a comprehensive investigation of Streptomyces is now indispensable.
Many authors have described the relevant mechanisms of Streptomyces using genomics, transcriptomics, proteomics, and so on, but there are hardly any metabolomics analyses which illuminate the metabolic mechanism of Streptomyces. Metabolomics is the best tool for understanding cell metabolism and the relationship between genotypes and phenotypes. Specifically, given improvements in mass spectrometry techniques, the field of metabolomics has experienced significant growth (Carnicer et al. 2012). However, while there have been many improvements in metabolomics, perfecting the procedure used to prepare biological samples is still a challenge. Many metabolites not only have a short turnover time which are just seconds or less, but they also have different physicochemical properties and extremely low intracellular concentrations (de Jonge et al. 2012; Douma et al. 2010). This means that in quantitative metabolomics studies, the key step is defining snapshots of all interesting metabolites. To this end, rapid sampling, fast quenching, and effective extraction procedures are crucial for ensuring the real status of intracellular metabolites. Due to the seconds or sub-seconds turnover time, keeping rapid sampling at a time of less than one second is reasonable. When quenching, it is essential that first, enzymes are inactivated immediately to ensure no loss or (inter)conversion of metabolites; second, that cells are kept intact to avert intracellular leakage; and third, that extracellular diffusion and exposure time are kept as short as possible to guard against the noxious effects of organic solvents. During the process of extraction, thorough, non-selective, and non-destructive operations are necessary.
Notably, Smart et al. developed an analytical platform for the metabolomic analysis of microbial cells (that is, yeast, filamentous fungi, and bacteria) using methyl-chloroformate (MCF) derivatization followed by GC–MS (Cheng et al. 2013). They demonstrated that using cold glycerol-saline solution as a quenching agent could stop the cell metabolism of different microbial cells (such as Streptomyces coelicolor) (Smart et al. 2010) while minimizing the leakage of intracellular compounds into the quenching solution, thus allowing the reliable separation of intracellular and extracellular metabolites (Villas-Bôas and Bruheim 2007). Specifically, cold glycerol saline appeared to have potential as a quenching solution for an accurate intracellular metabolite analysis based on MCF derivatization, but it had drawbacks as a result of silylation derivatization (Villas-Bôas and Bruheim 2007), which is the classic and most widely used derivatization procedure for metabolomics analysis by GC–MS (Villas-Boas et al. 2005). In addition, Kassama et al. assessed the extraction procedures of intracellular metabolites for Streptomyces lividans and quenched 5 mL of broth in 25 mL of 60% aqueous methanol which contained 10 mM HEPES at − 40 °C (Kassama et al. 2010). Wentzel and colleagues studied intracellular metabolite pool changes in Streptomyces coelicolor. In both of these studies, 5 mL of culture sample was withdrawn, filtrated, washed twice with 2.63% (w/v) NaCl solution, and transferred to 25 mL 60% methanol solution at − 23 °C (Wentzel et al. 2012). Unfortunately, the HEPES and NaCl solution had the potential to cause matrix effects on MS-based analysis, meaning that quenching should be finished as quickly as possible to obtain metabolism snapshots. While many efforts have been made to exploit the optimal method, no universal solution has been discovered for each individual micro-organism, meaning that it should be modulated individually.
Typical procedures of sample preparation for Streptomyces
Streptomyces albus (Lu et al. 2016)
9 mL sample
Acetonitrile/methanol/0.1% glacial acetate (45:45:10, v/v) to a final volume of 1 mL − 20 °C
Acetonitrile/methanol/0.1% glacial acetate (45:45:10, v/v); 1 mL; − 20 °C
Streptomyces avermitilis (Guo et al. 2015)
6 mL 0.6% (w/v) NaCl at 4 °C
4 g 40% methanol at − 40 °C
1 mL; chloroform: ethanol: water = 2:2:1 (v/v/v), thawed and frozen
Streptomyces coelicolor (Wentzel et al. 2012b)
5 mL sample
5 mL 2.63% (w/v) NaCl
25 mL 60% methanol at − 23 °C
25 mL 60% methanol at − 23 °C, thawed and frozen
Streptomyces hygroscopicus (Wang et al. 2015)
10 mL sample
10 mL 0.9% (w/v) NaCl at 4 °C
40 mL 60% methanol at − 40 °C
Grinded using liquid nitrogen; 1.0 mL cooled methanol (50% (v/v), − 40 °C); thawed and frozen
Streptomyces lividans (Muhamadali et al. 2015)
15 mL sample
30 mL 60% methanol at − 45 °C
0.5 mL 100% methanol at − 45 °C, thawed and frozen
Streptomyces lydicus (Cheng et al. 2013)
60% methanol at − 40 °C
Grinded using liquid nitrogen; cooled methanol (50% (v/v), − 40 °C)
Streptomyces tsukubaensis (Xia et al. 2013)
10 mL sample
10 mL 0.9% (w/v) NaCl
25 mL 60% methanol at − 40 °C
2.5 mL 50% (v/v) methanol at − 30 °C, thawed and frozen
Streptomyces lividans (Kassama et al. 2010)
5 mL sample
25 mL 60% methanol containing 10 mM HEPES at − 40 °C
1 mL 100% methanol at − 40 °C, thawed and frozen
In this area, the most popular procedure for sample pretreatment was a combination of cold methanol with boiling ethanol. However, it has been robustly shown that cold methanol quenching is always the cause of serious leakage of intracellular metabolites in bacteria (Min et al. 2010; Japelt et al. 2015; Link et al. 2008). In addition, the partial conversion of pyruvate, nucleotides, and phosphate sugars was observed during extraction with boiling ethanol (Winder et al. 2008). In bacteria, the fast filtration method was used and verified to be effective for minimizing the leakage of intracellular metabolites both in Gram-negative and Gram-positive bacteria (Hong et al. 2017; Bolten et al. 2007). This method can, therefore, be used as an alternative to cold methanol quenching. However, there has as yet been no systematic evaluation reported of this sample pretreatment method for Streptomyces ZYJ-6.
In this study, aiming to develop comprehensive tools for quantitative intracellular metabolomics of Streptomyces ZYJ-6, utilizing quenching methods and extraction procedures which have not been previously reported. By basing quenching solution on their molar transition energy (ET), we assessed the effects of five different solutions on the leakage of intracellular metabolites of Streptomyces ZYJ-6. To our knowledge, this is the first report of optimization quenching solution using molar transition energy (ET) and evaluating five quenching solutions and three extraction procedures (see Additional file 1: Table S1) through gas chromatography–isotope dilution mass spectrometry (GC–IDMS) for Streptomyces. We believe that our findings will be useful for the quantification of intracellular metabolites in other Streptomyces.
Materials and methods
Solvents and chemicals
Chemicals were provided by Shanghai Lingfeng (Chemical Reagent Co., Ltd., China), while analytical grade standards were purchased from Sigma-Aldrich Chemical Co. (USA) and LC–MS-grade solvents were obtained from Fisher Scientific (Thermo Fisher Scientific, USA).
Strain and cultivation
The Streptomyces ZYJ-6, a mutant strain with only a single component (FR-008-III) (Zhou et al. 2008), was kindly donated by professor Delin You of Shanghai Jiao Tong University, China.
Aerobic cultivations of Streptomyces ZYJ-6 were initiated with glycerol stocks. Spores were harvested from slant culture on SFM medium (2% agar, 2% mannitol, 2% soybean powder, and pH 7.2) after 4-day incubation at 30 °C. Spore suspension was inoculated (107 spores per 100 mL−1) in a 500 mL Erlenmeyer flask with 100 mL TSBY medium (3% TSB, 1% yeast extract, 10.3% sucrose, and pH 7.2) and grown for 30 h at 30 °C and 220 rpm. Some 300 mL of mycelia suspension was inoculated in 5 L bioreactor for additional fermentation.
Fermentation culture was carried out in a 5 L turbine-stirred bioreactor (working volume of 3 L) at 30 °C with agitation at 400 rpm. The sterile air was set at 1 vvm through a bottom sparger, maintaining overpressure at 0.05 MPa in chemically defined medium. The medium contained (L−1): glucose 50 g, KH2PO4 1.5 g (NH4)2SO4 1.8 g, EDTANa2 1.8 g, MgSO4·7H2O 8.6 g, ZnSO4·7H2O 35.7 mg, CaCl2 50 mg, FeSO4·7H2O 28.7 mg, CuSO4·5H2O 42 mg, MnSO4·H2O 9.1 mg, antifoam 0.3%, NaCl 9.0 g as an osmotic pressure regulator, and pH 7.2 was controlled by feeding 10% ammonium hydroxide. The bioreactor with medium was sterilized at 121 °C for 60 min, while the glucose solution was sterilized separately at 110 °C for 40 min.
Preparation of uniformly 13C-labeled cell extracts
Metabolite was accurately quantified by IDMS method (Wu et al. 2005). Extracts from cells grown on uniformly 13C labeled [U-13C] glucose were utilized as internal standards (IS) and added into each sample before extraction. IS was able to trace losses during sample pretreatment (Canelas et al. 2009). This was prepared by cultivating Pichia pastoris G/DSEL in 1 L turbine-stirred bioreactor (working volume 0.6 L) and was fed with fully U-13C (20 g/L, 99%, Cambridge Isotope Laboratories, Inc.) labeled glucose as the sole carbon source. Cultivation took 20 h (Hong et al. 2017; Lu et al. 2015). Sampling, quenching, and extraction procedures were carried out as described by Carnicer et al. (2012), although quenching solution was precooled and maintained at − 80 °C.
TB samples were immediately withdrawn into 15 mL centrifuge tubes containing 8 mL of quenching solution precooled at − 30 °C by rapid sampling device in 0.2 s, whereupon they were weighed and provisionally stored at − 30 °C. IC and QS samples were withdrawn with the same manner, although after weighing, they were centrifuged for 1 min at − 13 °C and 6300 RCF. Subsequently, samples were separated to pellet (IC) and supernatant (QS) and provisionally stored in cryostat at − 30 °C. Samples of TB, IC, and QS should then be subjected to extraction of the metabolites. If not, samples should be stored at − 80 °C until extraction. EC samples were withdrawn into a filter syringe precooled to − 20 °C with 32 g stainless steel beads (2 mm diameter, Shanghai Yalian Hardware Electromechanical Equipment Co., Ltd.), filtrated through a 0.45 µm PVDF filter (RephiLe Bioscience, Ltd.), and attached to a syringe. The filter liquor was then collected into new tubes, snap-frozen in liquid nitrogen, and stored at − 80 °C until MS analysis (Mashego et al. 2003).
Quenching and extraction
Quenching solutions in the experimental group were composed in two parts. Part A: Part B = 5:1 (v/v). Part A was the selected solution based on the different molar transition energy (ET, an empirical quantitative parameter of solvent polarity) (Reichardt and Christian 1979): acetone (ET = 42.2 kcal mol−1), isoamylol (ET = 47 kcal mol−1), propanol (ET = 50.7 kcal mol−1), and methanol (ET = 55.5 kcal mol−1), respectively. Part B, referred to as the base solution in this study, consisted of acetone (ET = 42.2 kcal mol−1):ethanol (ET = 51.9 kcal mol−1) = 1:1 (mol/mol). The quenching solution in the control group was 60% (v/v) methanol aqueous (water: ET = 63.1 kcal mol−1), as shown in Table 1. The above components were abbreviated as AceB, IsoaB, ProB, MethB, and Meth60, respectively. For example, AceB means that part A was acetone and mixed with part B by 5:1 (v/v). All other abbreviations follow this pattern. Meth60 was a 60% (v/v) methanol aqueous solution without the base solution. In the optimization experiments for quenching solutions, freezing–thawing in liquid nitrogen in 50% (v/v) methanol was used for extraction, following literature summarized in Table 1.
Extraction was optimized using three methods. First, after adding 100 μL IS to the samples (TB, IC, and QS), cell pellets/solutions were suspended/mixed with 30 mL 50% (v/v) methanol at − 30 °C. Samples were then subjected to three cycles of freezing in liquid nitrogen for 3 min and thawing at − 30 °C on cryostat. The second method followed the same steps as method one, but after being suspended/mixed, the cell pellets/solutions were then ground in a mortar with liquid nitrogen. Third, cell pellets/solutions were suspended/mixed with 30 mL of 75% (v/v) ethanol at 95 °C and the extracted by boiling for 4 min.
Procedures before injection
After extraction, 30–40 mL solution was centrifuged at 3580 RCF for 10 min at − 9 °C, while the supernatant was collected in a new tagged centrifuge tube and subjected to concentration. Once its weight was below 0.5 g, the concentrated solution was transferred to a new tube, where milliQ water was added to 0.5 g. After being filtrated with 0.22 μm filter, the 0.5 g concentrated solution was divided into four different aliquots. One and two were 100 μL*2 in gas chromatography (GC) vials, one for measurement of amino acids (AA) and the other for analysis of organic acids (OA), phosphate sugars (PS), and sugar alcohols (SA). The third was 50 μL in liquid chromatography (LC) vials for nucleotides and coenzymes determination, while the fourth was the remainder, stocked at − 80 °C for safety. An 80 μL EC sample was drawn into GC vials with 20 μL IS. This resulted in two aliquots: one for AA measurement and the other for analysis of OA, PS, and SA. A 40 μL EC sample was drawn into LC vials with 10 μL IS for determination of nucleotides and coenzymes.
Samples in GC vials were lyophilized and derivatized. For the AA analysis, 75 μL of acetonitrile and 75 μL of N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) were added to the samples. The resultant samples were incubated at 70 °C for 60 min (de Jonge et al. 2011). For analysis of OA, PS, and SA, 50 μL of fresh pyridine methoxamine solution (20 mg mL−1) were added to the GC glass vials. These vials were then incubated at 70 °C for 50 min. After being cooled to room temperature, 80 μL methyl-trimethyl-silyl-trifluoroacetamide (MSTFA) was added, and the samples were then incubated at 70 °C for 50 min (Cipollina et al. 2009). Before injection, samples were centrifuged in 8000 RCF for 1 min, following which the supernatant was transferred to an inner liner.
AA, OA, PS, and SA were analyzed using GC–MS with a 7890A GC paired with a 5975C MSD (Agilent, Santa Clara, CA, USA). Conditions for the determination of the metabolites were as defined by de Jonge et al. 2011, with slight modifications to column and temperature gradients. Briefly, 1 µL of sample was injected on a HP5-5% Phenyl Methicone column (30 m × 250 µm internal diameter and 0.25 µm film thickness), using 250 °C for injection in splitless mode. During analysis, the flow velocity of helium was set as 1 mL min−1. The GC column temperature gradient for AA analysis was initially set to 100 °C for 1 min and was then raised by a speed of 10 °C min−1 up to 300 °C and held at 300 °C for 10 min. In contrast for OA, PS, and SA, initial temperature was set as 70 °C for 1 min and then increased with a speed of 10 °C min−1 up to 300 °C and kept at 300 °C for 10 min. The temperature of transfer line, MS source, and quadrupole were set as 280 °C, 230 °C, and 150 °C, respectively. Electron ionization was always operated with 70 eV. To ensure accurate quantitation, MS was operated in selected ion-monitoring (SIM) mode. This means that the results reported in this study refer to quantitative targeted metabolomics experiments outside of untargeted metabolomics experiments. The quantification of metabolites was conducted using IDMS (Wordofa et al. 2017), while the unit of metabolites concentration was μmol gDCW−1.
Nucleotides and coenzymes were analyzed utilizing an ultra-high-performance liquid chromatography–mass, tandem–mass spectrometry (UHPLC-MS/MS). The Thermal Ultimate 3000 UPLC system was coupled to a Thermal TSQ QUANTUM ULTRA mass spectrum system. Specifically, separation of compounds was conducted by an ion-pairing reverse-phase method on an ACQUITY UPLC BEH C18, 1.7 μm, 150 × 2.1 mm column (Waters Corporation, Milford, MA, US) at 25 °C. The eluent A was 5% acetonitrile aqueous solution with 5 mM dibutylammonium acetate (DBAA), while the eluent B was 84% acetonitrile aqueous solution with 5 mM DBAA. The gradient of eluents A and B was used as defined by Seifar et al. (Seifar et al. 2013, 2009) (Additional file 1: Tables S2 and S3). The injection volume was 2 µL. MS was operated in selected reaction monitoring (SRM) with a negative ion mode. Electrospray ionization parameters were as follows: spray voltage 3000 V, sheath gas pressure 15 arbitrary units, aux gas pressure 10 arbitrary units, ion sweep gas pressure two arbitrary units, capillary temperature 270 °C, and vaporizer temperature 200 °C. Daughter ions, tube lens voltage, and collision energy were optimized individually for each of these compounds (Hong et al. 2017).
The raw data of the chromatogram from GC–MS and LC–MS/MS were converted into concentration data using Chemstation from Agilent and Xcalibur from Thermo Fisher, respectively. The calibration curve was defined via IDMS (X-axis was the concentration of standard and Y-axis was the area ratio of 12C/13C). Following this, intracellular metabolites data were calculated according to μmol per dry cell weight.
Results and discussion
Batch cultivation of Streptomyces ZYJ-6
The suitability of AceB, IsoaB, ProB, MethB, and Meth60 were tested for the metabolomics analysis of Streptomyces ZYJ-6, based on the impacts of ET, mass balance analysis, and prolonged exposure time. The temperature in the quenching experiment process was maintained at below − 20 °C to inactivate metabolism. In all optimized approaches, three samples were obtained at a stationary state for biological replicates. In addition, the ET used in this study ranked from large to small successively were water, methanol, ethanol, propanol, isoamylol, and acetone. It was found that Meth60 possesses higher ET, while methanol was a small molecule and more liable to permeate the cells. This was probably the main reason for the leakage of intracellular metabolites of bacteria. Therefore, this study also considered the ET of the base solution. This base solution consisted of acetone and ethanol, the weakest and strongest ET combination. Ethanol has a larger molecule and stronger ET, which makes it a good candidate for a base solution for balancing the ET of the entire quenching solution.
Effect of E T
The 60% methanol and its derivatives (including some buffers) (Kapoore et al. 2017) have been widely used as a quenching solution for micro-organisms including Penicillium chrysogenum (de Jonge et al. 2012), Aspergillus niger (Lameiras et al. 2015), Pichia pastoris (Carnicer et al. 2012), and others. Despite this, more studies have verified that 60% methanol was a threat to the integrity of the prokaryotic cell membrane (Japelt et al. 2015; Link et al. 2008). Based on this, we hypothesized that the leakage might be related to the molecular size and ET value of quenching solutions. We, therefore, evaluated the effects of four combinations of quenching solutions from appropriate molecular sizes, and ET levels by quenching replicate samples in AceB, IsoaB, ProB, MethB, and Meth60, as well as comparing the concentration of the metabolites measured in the different fractions (Fig. 1).
The results in Fig. 3 and Additional file 2: Figure S2 show that in both QS and IC, the IsoaB with smaller ET was the best quenching solution, while the AceB with the smallest ET had the poorer performance. It could be speculated that the appropriate ET for each quenching solution was needed to avoid leakage. In addition, the MethB (~ 83% methanol), which was even lower than Meth60 (60% methanol) in the IC areas, serves as a reminder that methanol as a small molecule was threatening for Streptomyces ZYJ-6. Thus, results show that as a key parameter in selection of quenching solutions, ET had its optimal range. In terms of Streptomyces ZYJ-6, the best quenching condition was the IsoaB at − 30 °C.
Mass balance analysis
The extent of the leakage of metabolites from quenching in AceB, IsoaB, ProB, MethB, and Meth60 was assessed using a quantitative mass balance method (Lameiras et al. 2015). To compare the performance of the five quenching solutions, the ratio of metabolite concentration in IC as well as the difference between the concentrations in TB and EC were calculated for each metabolite from each of the quenching solutions. These ratios can be seen as types of recoveries, namely recovery ratio = IC/(TB − EC) × 100%. It has been stated in the literature (Bolten et al. 2007; Taymaz-Nikerel et al. 2009) that the subtraction procedure is widely accepted for estimating the ‘true’ intracellular amount.
It was concluded that using IsoaB as the quenching solution at − 30 °C resulted in the best performance among the five quenching candidates. However, for many metabolites, especially organic acids, this combination was unable to completely prevent leakage.
Impact of prolonged exposure
Much existing research has reported the impact of prolonged exposure in quenching solutions. Koning et al. stopped the leakage of prolonged exposure in the methanol quenching solution by measuring metabolite levels after 30 min of quenching (Koning and Dam 1992). However, results from de Jonge et al. and Canelas et al. demonstrate that prolonging exposure time in quenching methanol solutions aggravates the leakage of intracellular metabolites (de Jonge et al. 2012; Canelas et al. 2008). Our experiment was implemented by keeping samples with IsoaB in the cryostat at − 30 °C for + 0, + 5, or + 30 min, respectively before the centrifugation step. We anticipated that the same intracellular levels would be found regardless of exposure time if leakage did not take place. As shown in Fig. 5b, fumarate (Fum) and trehalose-6-phosphate (T6P) were typical examples of organic acids (smaller molecule, less polar) and phosphorylated intermediates (larger molecule, more polar), respectively. It is clear that the longer the cells remained in the quenching solution, the larger were the error bars gained, indicating some extent of time-dependent leakage occurring in intracellular metabolites. Interestingly, T6P, which acted as the larger and more polar compound, exhibited a lesser extent of leakage than the smaller and less polar compounds, such as Fum. These results were broadly in agreement with the existing findings (Canelas et al. 2008), suggesting that contact time with the quenching solution should be kept to a minimum, as leakage was not serious in this case.
Evaluation of extraction procedures
In contrast, BE could overcome the aforementioned disadvantages to a certain degree. However, some existing studies have reported that several metabolites (pyruvate, nucleotides, and sugar phosphates) (Winder et al. 2008; Maharjan and Ferenci 2003; Villasbôas 2005) were not stable in 95 °C 75% (v/v) ethanol, which could be a fatal defect in the extraction of those metabolites. Grinding (G) in liquid nitrogen within 50% (v/v) methanol had some differences across different metabolites, while it is easy to cause experimental human errors and loss of materials during the grinding process. In summary, in the laboratory scale and low-throughput condition, thawing–freezing (TF) in cryostat at − 30 °C as well as liquid nitrogen within 50% (v/v) methanol for three cycles was found to be an appropriate extraction procedure for the intracellular extraction of Streptomyces ZYJ-6.
Composition of the Streptomyces ZYJ-6 metabolomics
In total, 44 intracellular metabolites were found and quantified from AA, OA, PS, SA, nucleotides, and coenzymes, based on IsoaB and Meth60 as the quenching solutions and thawing-freezing (TF) in cryostat at − 30 °C and liquid nitrogen in 50% (v/v) methanol for three cycles as the extraction solution. The 44 intracellular metabolites were identified and confirmed using the standards in quantitative targeted metabolomics experiments. Results are displayed in Additional file 1: Table S5. While these metabolites could not represent the entire metabolomics of Streptomyces ZYJ-6, they did cover the most highly abundant metabolites, which played significant roles in central, amino acid and even energy metabolism. The ratio of intracellular metabolites concentrations in IsoaB/Meth60 was calculated and can be seen in Additional file 1: Table S5, which was approximately 2–10, suggesting that the largest concentrations of intracellular metabolites in IsoaB were higher than those in the traditional 60% (v/v) methanol quenching solution. Inspection of Fig. 6b suggests that almost all black squares (intracellular metabolites from IsoaB quenching) were above red polygons (intracellular metabolites from Meth60 quenching), meaning that 60% (v/v) methanol quenching solution may cause severe leakage, and so the IsoaB was the optimal quenching solution for Streptomyces ZYJ-6.
First of all, based on the effect of ET, mass balance analysis, the impact of prolonged exposure, and the quantitative determination of metabolites, we found that isoamylol with a base solution as the quenching solution and thawing–freezing as the extraction procedure were the most reliable, accurate, and reproducible sample pretreatment method to meet our aim. Furthermore, ET value was an important parameter for choosing the quenching solutions for different micro-organisms. Finally, we demonstrated 44 identified and quantified intracellular metabolites in Streptomyces ZYJ-6, which was unprecedented and, therefore, important in the study of metabolomics. Our work is the first to shed light on the tools for quantitative intracellular metabolomics of Streptomyces ZYJ-6 and has the potential for significant influence in the relevant fields, such as 13C-based metabolomics flux analysis and multi-omic research as well as genome-scale metabolic model establishment. In addition, this work provided important evidence for research into additional Streptomyces.
We thank Prof. Dr. Delin You (Shanghai Jiao Tong University) for supplying Streptomyces ZYJ-6.
XL, TW, and XS carried out experiments, and XL was the major contributor in writing the manuscript. ZW, XT, YZ, and JC revised manuscript. All authors read and approved the final manuscript.
This work was financially supported by a grant from the Major State Basic Research Development Program of China (973 Program, No. 2012CB721000G), NWO-MoST Joint Program (2013DFG32630), and National Key Special Program 2017YFF 0204600.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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