3. 1 Define the optimal quenching and extraction procedure for
quantitative metabolomics
Accurate determination of intracellular metabolite levels requires
well-established procedures for sampling and sample pretreatment. The
ideal procedure should immediately quench cellular metabolism and
quantitatively extract all metabolites, while a significant challenge
associated with the rapid turnover, varied abundance and physicochemical
diversity of intracellular metabolites still requires systematic and
quantitative evaluation. Otherwise, the obtained biased metabolite level
will lead to misinterpretation of functional information about the
biochemical and physiological states of cells [40]. In recent years,
different quenching and extraction methods have been proposed and used
for quantitative metabolomics of mammalian cells including tumor cells
(Table 1 ). Early quenching studies on microorganisms
established the use of cold methanol as a standard quenching protocol
for its high efficiency of freezing enzyme activities [43-45], and
cold methanol has also been reported frequently as the quenching agent
in mammalian cell metabolomics [20, 24, 46, 47]. In addition to cold
methanol, the use of cold normal saline has also become common and cold
0.9% (w/v) NaCl has been preferably used for quenching mammalian cell
metabolism with minimal metabolite leakage [48]. Meanwhile, liquid
nitrogen was also a commonly used quenching method in recent years which
could rapidly and efficiently stop cell metabolism [18, 49-52].
After cell harvesting and quenching, metabolite extraction was the next
critical step. Several methods existed for metabolite extraction, but
the literatures were sometimes contradictory regarding the adequacy and
performance of each technique [41]. To the best of our knowledge,
the polarity of metabolites ranged from very hydrophilic to hydrophobic
compounds, and polar metabolites were supposed to be extracted with
ethanol, methanol, acetonitrile, or other mixtures of these solvents,
etc [18, 24, 26, 46, 48, 51, 52], while more lipophilic metabolites
could be extracted by chloroform/methanol/water mixture, etc [20, 47,
49]. At the same time, in order to ensure that the extraction of
intracellular metabolites was as complete as possible, the extraction
was usually combined with several freeze-thaw cycles [18].
However, no previous study has yet simultaneously optimized these
critical procedures, i.e., quenching and extraction, to explore cell
metabolomics using a strictly quantitative approach. Therefore, in this
study, we systematically evaluated 12 combinations of three commonly
used quenchers and four extractants using an IDMS approach and the
specific experimental workflow were shown in Figure 1 . The
prepared samples were analyzed using gas/liquid chromatography-IDMS
method, and a total of 43 metabolites were quantitatively determined in
Hela cells, including amino acids, sugar phosphates, organic acids,
adenosine nucleotides and coenzymes (Figure 2 ).
As shown in Figure 1 , the medium was first removed and the
cells attached to the flask surface were washed twice with PBS. The
conventional method generally used trypsin to detach cells from their
growth surface, which has been proven to inevitably change the profile
of cellular metabolites since the detachment of cells from the
extracellular matrix altered their physiology [18, 53-55]. In order
to avoid changing the cellular environment before quenching metabolic
activities within the cell, in this study, tumor cells cultivated in 2D
mode were directly quenched and then scraped with the extraction solvent
in all experiments. Meanwhile, 3D MTSs were first digested into single
cell suspension, and the cell pellet was obtained after centrifugation.
The subsequent operations were the same as those of the 2D cells. We
estimated the cell number by determining the protein content of cell
pellet. The standard curve of the protein was shown in Figure
S1 . With respect to quenching efficiency, ideal quenching solvent
should immediately inhibit cellular metabolic activity and inactivate
enzymes within the cells without leaking metabolites from the cells.
Quenching of metabolism was usually achieved by rapid changes in
temperature or pH. The rate of quenching was critical because many
metabolic reactions in glycolysis, as well as those related to ATP,
featured high turnover rates in the timescale of seconds [45].
Numerous studies have reported the adverse effects of prolonged exposure
to quenching solutions which demonstrated that the longer the cells were
exposed to the quenching solution, the exacerbated leakage of
intracellular metabolites [22, 45, 56]. This seemed reasonable that
prolonged exposure to organic solvents and cold shock could cause cell
membrane leakage, which also implied that sample processing should
proceed as quickly as possible to the extraction step. In addition,
quantitative methods should be used to determine the extent of cellular
leakage so that true intracellular metabolite concentrations could be
determined [45]. To achieve this, we quantitatively assessed the
number and amount of leaked metabolites in the quenching agent, and
investigated the factors that influenced the extent of leaked
metabolites.
In order to determine the most effective extraction method, four
different extraction methods were evaluated after Hela cells were
quenched with liquid nitrogen, 50% methanol, and normal saline
respectively. The evaluated extraction methods included 80% methanol,
50% acetonitrile, methanol: chloroform: water (1:1:1, v/v/v) and 70%
ethanol (75℃). Sellick et al. found that the addition of a water-based
extraction step combined the properties of solvent-based and water-based
methods, and extracts produced in this method showed the greatest amount
of recovered metabolites in CHO cell [26]. Therefore, we also
adopted an additional water extraction to increase metabolite coverage
for all 12 combination methods. The metabolites in the obtained cell
extracts were absolutely quantified using GC-IDMS and LC-MS/MS-IDMS
method. Subsequently, effectiveness of the quenching and extraction
procedures should be quantitatively evaluated for their suitability for
Hela carcinoma cells grown as 2D monolayer cultures and 3D MTS. For this
purpose, previous studies have reported some evaluation criteria with
respect to relative metabolite abundance and metabolite coverage. For
example, LORENZ et al. established a method for relative quantification
of metabolites in adherent mammalian cells using the clonal β-cell line
INS-1 as a model sample, without considering absolute quantification of
metabolites [49]. Dietmair et al. evaluated several quenching and
extraction procedures for mammalian cells grown in suspension, but did
not take into account the reproducibility of each method, with large
errors in the metabolite level [48].
To allow an accurate view of intracellular metabolome, the quenching
step should stop metabolism as quickly as possible while the extraction
should cover a wide spectrum of metabolite. Furthermore, target
metabolites should not undergo any physical or chemical modification and
degradation should be minimized during quenching and extraction process
[45]. In this study, we evaluated the compatibility of three
different quenchers such as liquid nitrogen, -40°C 50% methanol, 0.5°C
normal saline and four extractants (80% methanol, methanol: chloroform:
water (1:1:1, v/v/v), 50% acetonitrile, 75°C 70% ethanol) for global
metabolite profiling of adherent Hela carcinoma cells. To
comprehensively evaluate 12 combinations of quenching and extraction
procedures in this study, we analyzed the acquired metabolite data based
on the following four criteria: 1) Metabolite leakage in the quenching
solution; 2) Metabolite coverage and abundance identified in cell
extracts; 3) Absolute quantitation of intracellular metabolite
concentrations; 4) Reproducibility of metabolite quantification
[57].
3.1.1 Metabolite leakage in the
quenching solution
To study the effect of quenching solutions on the integrity of cell
membrane, the potential metabolite leakage during quenching was
evaluated by quantifying the metabolites in the quenching solution. The
metabolites analyzed include compounds with different properties, such
as amino acids, organic acids, sugar phosphates, adenosine nucleotides
and coenzymes. Figure 3 showed the effect of these two
quenchers (50% methanol and normal saline) on the recovery of different
representative metabolites. Liquid nitrogen did not cause intracellular
metabolites leak into the quencher because liquid nitrogen would
volatilize directly. In both cases, all determined amino acids were
found in the quenching solution. Compared with normal saline, cold
methanol gives rise to more serious leakage of intracellular amino acids
(Figure 3a ). Also, an obvious phenomenon showed that the more
leakage comes with the higher amounts of intracellular amino acids, such
as alanine (Ala), valine (Val), proline (Pro), lysine (Lys), aspartate
(Asp), glutamate (Glu). In consistency with amino acids, organic acids,
sugar phosphates and energy metabolism intermediates also exhibited a
significant higher leakage when tumor cells were quenched with the cold
methanol. Meanwhile, the results also indicated that intracellular sugar
phosphates were more inclined to leak in response to cold methanol
treatment (Figure 3b ). According to previous reports, the
higher degree of leakage caused by cold methanol may be partly
attributed to the following aspects. The first was the increase in cell
membrane fluidity or the decrease in thickness during the quenching
process [57]; The sudden increase in cell membrane permeability
caused by ”cold shock” was also reported [58]; In addition, methanol
was a small molecule which was easier to penetrate cells which may also
be the reason for the leakage of intracellular metabolites [22].
Moreover, Canelas et al. found that the extent of metabolite leakage inS. cerevisiae was affected by the methanol content of the
quencher, and reducing the methanol concentration resulted in the
increase of intracellular metabolite leakage [44]. Therefore, the
necessity of fine-tuning quenching strategy should be emphasized. In
general, it could be concluded that methanol quenching was not suitable
for accurate quantification of the metabolites in Hela cells. Normal
saline was the better of the two quenchers, although it could not
completely prevent leakage.
Furthermore, we investigated the factors such as the molecular weight
which has been reported to affect the degree of metabolite leakage
[56]. Figure 4 showed the relationship between the degree
of metabolite leakage and molecular size which was represented by
molecular weight. We found that metabolites with a smaller molecular
weight were more seriously leaked than metabolites with a larger
molecular weight when normal saline was used as the quencher. This was
consistent with the findings by Canelas et al. observed in S.
cerevisiae that the leakage of the metabolites into the quenching
solution was mostly driven by diffusion [56] and the diffusion rate
of smaller molecules was higher [44]. In contrast with this, when
cold methanol was used as the quencher, the degree of metabolite leakage
showed no clear relation with the molecular weight, and severe leakage
was observed to be widespread in the detected metabolites.
3.1.2 Metabolite coverage and abundance identified in cell
extracts
The comparison of quantitative whole-cell metabolites showed that the
number and total amount of metabolites extracted under each condition
were significantly different (Figures 5 and 6) . For example,
Oxaloacetic acid (OAA) was below the detection limit with
methanol/chloroform as the extractant, but detectable in all the other
extractants. Most sample preparation procedures frequently used cold
organic solvents to stop cellular metabolic activities. In this study,
we also quenched cellular metabolism directly by adding liquid
N2 on the flask plate, followed by the addition of
extraction solvent. Our results showed relatively less metabolites were
obtained with methanol as the quencher compared to cells quenched with
normal saline or directly quenched with liquid N2.
Kapoore et al. also found that methanol quenching treatments resulted in
severe leakage of nearly all metabolite classes in metastatic breast
cancer cell line MDA-MB-231 [29]. In addition to this, the result
also showed that direct quenching with liquid N2produced the highest recovery of all metabolites compared with methanol
and normal saline (Figure 6 ). Additionally, the overall effect
of normal saline as the quenching agent was between liquid nitrogen and
methanol. Consistently, one previous study also reported that quenching
with 60% methanol (buffered or unbuffered) resulted in leakage of
intracellular metabolites from the cells. Whereas, quenching with cold
normal saline (0.9% [w/v] NaCl, 0.5℃) did not damage the cells and
effectively prevented the conversion of ATP to adenosine diphosphate
(ADP) and adenosine monophosphate (AMP), indicating metabolic arrest in
CHO cells [48].
Furthermore, based on the number of metabolites extracted, acetonitrile
shows the best extraction efficiency (Figure 5 ). In addition to
the total number of extracted metabolites, the total amount of extracted
metabolites was also an important indicator for evaluating the
extraction methods. The results showed that using liquid nitrogen as the
quencher and 50% acetonitrile as the extractant, the maximum total
amount of intracellular metabolites could be harvested, about 295
nmol/million cells, which was 1.67 times to 13.73 times higher than
other combination methods. In addition, it could be observed that this
optimal method allowed a high extraction capacity for adenosine
nucleotides and coenzymes (Figure 6 ). For the four different
extractants, it can be inferred that the extraction efficiency of
acetonitrile and ethanol was generally higher, while the extraction
efficiency of methanol: chloroform: water (1:1:1, v/v/v) (M/C) was the
worst regardless of the type of quenchers (Figure 6 ).