June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Research Paper

Inﬂuence of matrix eﬀects by inorganic ions insingle-cell direct mass spectrometry
1

2

1

3

4

Yoshihiro Kato , Shinobu Kudoh , Eiji Sugiyama , Iwao Sakane , Naohiro Tsuyama ,11
1,5
Toshimasa Toyoʼoka , Kenichiro Todoroki , Hajime Mizuno *Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences,
University of Shizuoka, 52‒1 Yada, Suruga-ku, Shizuoka, Shizuoka 422‒8526, Japan
2
Yokogawa Electric Corporation, 2‒9‒32 Nakacho, Musashino, Tokyo 180‒8750, Japan
3
Central Research Institute, ITO EN, Ltd., 21 Mekami, Makinohara, Shizuoka 421‒0516, Japan
4
Department of Radiation Life Sciences, Fukushima Medical University School of Medicine,
1 Hikarigaoka, Fukushima, Fukushima 960‒1295, Japan
5
Faculty of Pharmacy, Meijo University,
150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468‒8503, Japan
1

Abstract Direct nanoelectrospray ionization mass spectrometry (nanoESI-MS) is extremely useful for single-cell analysis
to detect endogenous metabolites as well as exogenous compounds that have been taken up. However, during ionization,
coexisting constituents, especially sodium chloride, can cause severe matrix eﬀects, resulting in decreased detection sensitivity and ﬂuctuating peak intensities for the compounds of interest. Herein, conventional ESI and nanoESI were examined
to determine the extent of ionization suppression by salts using amino acids as typical cellular constituents in addition to
caﬀeine and acetaminophen as exogenous compounds. The corresponding isotope-labeled standard compounds were also
tested to compensate for ﬂuctuations. All the tested compounds showed signiﬁcantly decreased ion intensities with bothconventional ESI and nanoESI when ionized with high-concentration salt solutions. However, when the nanospray tip withthe smallest inner diameter was used for nanoESI, the peak intensities were 2‒3 times higher than those obtained using
conventional ESI and their peak intensities recovered to 60% compared to the salt-free samples. These ﬁndings clearly
demonstrate that nanoESI has advantages over conventional ESI in terms of reducing ionization suppression eﬀects forsmall molecules in biological samples such as cells containing inorganic salts, particularly sodium chloride, at high concentrations. Furthermore, the use of stable isotope-labeled compounds corresponding to each analyte was revealed to allow
adequate correction of the ion peak intensity ﬂuctuations for each analyte during direct nanoESI-MS analysis of single
HepG2 cell samples, even when coexisting salts cause severe ion suppression.
Key words: single-cell analysis, direct infusion mass spectrometry, nanoelectrospray ionization, matrix eﬀect, amino acid

Introduction

research group for the rapid analysis of cellular phenomena.

Live single-cell MS was previously developed by ourIn this method, the cellular components in a single living cellwere captured into a metal-coated glass capillary under a*Corresponding author

microscope, and the extracted cellular molecules were1)
directly analyzed by nano electrospray (nanoESI)-MS .

Hajime Mizuno

Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468‒8503, Japan

Using direct nanoESI-MS, it has become possible to detect
trace samples at the single-cell level without sample loss

Tel: ＋81‒52‒839‒2720
E-mail: hmizuno@meijo-u.ac.jp

through absorption by an LC column. This method has

Epub April 6, 2023.

been successfully used to develop various applications such1)
as intracellular organelle metabolism analysis , drug sub-

DOI: 10.24508/mms.2023.06.003

cellular localization analysis

Received: January 31, 2023. Accepted: February 21, 2023.

2‒4)

43

5‒8)

, and plant cell analysis.

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Recently, for high-throughput biomolecule analysis,
direct infusion MS without LC separation has been devel-

13
taining C-labeled metabolites (alanine, leucine, phenylala-nine, tryptophan, tyrosine, caﬀeine, glucose, benzoate,

oped using a high-resolution mass spectrometer . Shotgun
lipidomics is a direct MS method for analyzing lipid mole-citrate, octanoate, propionate, stearic acid, succinic acid,and sucrose) was purchased from Cambridge Isotope Labo-cules that are extracted from biological samples without LC

ratories (Tewksbury, MA, USA). Lyophilized human serum

9)

10)

separation . The use of nanoelectrospray ionization

was purchased from Nihon Pharmaceutical (Tokyo, Japan).
LC/MS-grade methanol was purchased from Kanto Chemi-(nanoESI) enables the highly sensitive mass spectrometric11)
detection of trace samples . In addition, data-independent
can be used to identify the detected ion peaks.

cal Co. (Tokyo, Japan). LC/MS-grade acetonitrile, formic
acid, and ammonium acetate (Optima LC/MS) were pur-Ion mobility MS can separate lipid ions with similar m/z val-chased from Fisher Scientiﬁc (Waltham, MA, USA). All

ues depending on diﬀerences in lipid structure and identify

other reagents and solvents were of analytical reagent

12,13)

acquisition

14,15)

the corresponding peaks, and can also identify chiral

grade. Water was puriﬁed using a PURELAB ﬂex ultrapure

16)

metabolites when combined with chiral derivatization .

waterHowever, as no chromatographic separation is performed
in direct MS, coexisting components in the sample can sup-Wycombe, UK).

puriﬁcation

system(ELGA

LabWater,High

Sample Preparationpress or enhance the ionization of analytes. This matrix
eﬀect, which causes ﬂuctuations in peak intensities between

High-concentration salt-spiked samples

samples and ion suppression, results in reduced detection. During the direct MS

In this study, amino acids were used as endogenous
metabolites, and caﬀeine was used as an exogenous sub-analysis of biological samples, high concentrations of salts

stance model, respectively. For each analyte, stock solu-

17,18)

sensitivity and reproducibility

such as sodium, potassium, and chloride have a very large

tions with various concentrations (10 μm, 1 μm, 100 nM,

19,20). In our previous study, it

and 10 nM) were prepared in saline (154 mmol/L NaCl

was reported that the ionization suppression eﬀect of salt

solution). Each stock solution was further diluted 10 times

was greater than that of cell extracts and serum in direct

with 80% methanol containing 0.1% formic acid to obtaininﬂuence of ion suppression

4)

nanoESI analysis .

concentrations of 1 μm, 100 nM, 10 nM, and 1 nM. The ﬁnal

In this study, we demonstrated the eﬀects of salts which

NaCl concentration of each sample was 15.4 mmol/L. The
C-labeled internal standards from the metabolomics QCfrom medium and cell lysates on metabolite analysis bydirect single-cell MS for accurate comparative analysis of13

metabolites expressed in each cell. To reduce the matrix

1 μm. Samples containing other salts (154 mmol/L KCl,
154 mmol/L LiCl, and 154 mmol/L NaH2PO4) were pre-kit were added to the ﬁnal samples at a concentration ofeﬀect caused by salts, we decreased the inner diameter ofnanoESI emitter and introduced normalization by iso-pared in the same way. The control samples were preparedtope-labeled internal standards. This detailed data on salt

by dissolution in water with no added salts.

eﬀects during direct nanoESI are expected to be useful for
improving the sensitivity and reproducibility of direct MS
for the analysis of metabolites in single-cell samples.

Cultured cells and sample collection

Finally, single-cell mass spectrometry using HepG2 cells

Osaka, Japan) was incubated in a CO2 incubator (5% CO2,37°C) with Dulbecco's modiﬁed Eagleʼs medium (FUJIF-

The human hepatoma cell line (HepG2, JCRB Cell Bank,

was performed to verify the normalization of the data by

ILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (biosera, EU ori-

the internal standard.

Experimental
Materials

gin, FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), 1% of Gibco 100 × MEM non-essential amino acidssine), caﬀeine, and acetaminophen were purchased from

(Life Technologies Corp., Gland Island, NY, USA), and 1%
of Gibco 100 × Antibiotic-Antimycotic (Life Technologies

FUJIFILM Wako Pure Chemical Corporation (Osaka,

Corp., Gland Island, NY, USA). Before sampling, the cells

Japan). Citric acid was purchased from Tokyo Chemical
Industry Co. (Tokyo, Japan). A metabolomics QC kit con-were washed three times with PBS (−) (Nacalai tesque,Amino acids (L-phenylalanine, L-tryptophan, and L-tyro-

Kyoto, Japan) and the medium was exchanged with PBS
44

June 2023

Medical Mass Spectrometry Vol. 7 No. 1(−). The HepG2 cells were observed with an inverted ﬂuorescence microscope (IX81, Olympus, Japan). The cyto-

Table 1. SRM transitions of analytes and their isotopelabeled internal standards

plasm of the target cells was directly micro-sucked into a
nanospray tip (Cellomics Tip CT-1, 3 μm of tip inter diame-

CompoundCollisionenergy (V)Q1 (m/z)

Q3 (m/z)

Leucine13
C- Leucine

132.1
138.1

86.1
91.1

25

Phenylalanine
13
C-Phenylalanine

166.1
1372.1

120.1
126.1

25

Tyrosine
13
C-Tyrosine

182.1
188.1

136.1
142.1

25

Tryptophan
13
C-Tryptophan

205.1
216.1

146.1
153.0

30

Acetaminophen

152.1

110.1

35

(tip approximate bore size: 3 μm), and Cellomics Tip CT-10(tip approximate bore size: 10 μm), HUMANIX, Hiro-Caﬀeine

195.1

138.1

30

shima, Japan), and the tip was set on a nanoESI ion source.

USA). The mass spectrometric conditions were as follows:

Full scan mass spectrometric detection was performed on a
Q-TOF mass spectrometer (Xevo G2-XS Q-Tof, Waters,

maximum injection time, 100 ms; mass resolution, 120,000
at m/z 400; scan range, m/z 100‒700; ion transfer tube tem-

Milford, MA, USA). The spray voltages for positive andnegative ion detection were 1.0 kV and −0.8 kV, respec-

perature, 200°
C; ion spray voltage, 1000 V for positive ion

ter, HUMANIX, Japan) with a micro-manipulator (MHW-3,
Narishige, Japan) operation, respectively. After sampling,
3 μL of ionization solvent (80% methanol containing with
0.1% formic acid and metabolomic QC kit solution) was
added into the nanospray tip from the back of the tip.

MS Conditions
nanoESI high resolution MS for salt spiked sample

A 3 μL salt spiked sample was added to the metal-coatedglass capillary nanoelectrospray tips (Cellomics Tip CT-1

mode.

tively. The mass spectrometric conditions were as follows:

Data Analysis

cone voltage, 20 V; source temperature, 100°
C; cone gas

The MS data were evaluated using the MassLynx

ﬂow, 0 L/h; nano ﬂow gas, 0.20 bar; purge gas ﬂow, 350 L/h;mass range, m/z 70‒1,000; analyzer mode, sensitive; scan

(Waters, Milford, MA, USA) and the Xcalibur (Thermo

time, 0.5 s/scan; data format, continuum; acquisition time,
3.0 min. The mass spectrometer was calibrated before analy-

Fisher Scientiﬁc, MA, USA) software and exported to a

sis using a calibration solution containing sodium formate.

list was performed using the MarkerView software (Sciex,
Framingham, MA, USA). Statistical analysis and calibra-

nanoESI QQQ MS for salt spiked sample

tion curve preparation for each substance were performed

peak list. Peak alignment for the peak list of each sample

A 3 μL salt spiked sample was added to the nanoelectro-

using Excel (Microsoft).

spray tip, and the tip was set on a nanoESI ion source.
Selected reaction monitoring (SRM) detection was per-Results and Discussion

Analysis of high salt samples by direct nanoESI-MS

formed on a QQQ mass spectrometer (Xevo TQ-S micro,

To demonstrate ion suppression eﬀects by salts, standard
samples of the analytes (1 μm in 15.4 mM NaCl) were ana-

Waters, Milford, MA, USA). The spray voltage was set at
1.0 kV for positive ion detection. The mass spectrometricconditions were as follows: cone voltage, 20 V; source tem-lyzed by direct infusion nanoESI high resolution MS using

perature, 100°
C; cone gas ﬂow, 0 L/h; nano ﬂow gas, 0.20
bar; purge gas ﬂow, 350 L/h; data format, continuum; acqui-

3 μL of the sample solution in a nanoESI tip (tip i.d. 3 μm).

sition time, 3.0 min. The SRM transitions of the analytes

standard samples with and without salt (NaCl and KCl) atare shown in Table 1.

the same concentration. In the expanded spectra of eachFig. 1 shows the high resolution full scan mass spectra ofanalyte (phenylalanine, tyrosine, tryptophan, and caﬀeine),

NanoESI high resolution MS for single-cell sample

the peak intensities of them in the sample decreased withsalt addition. In the LiCl-spiked sample, none of the ion

spectrometer (Orbitrap Fusion Lumos Tribrid equippedpeaks of these analytes were detected.
Fig. 2(a)‒(d) show the average peak intensities (n＝3) of

For single-cell analysis, we used a high-resolution mass

with a nanoESI source, Thermo Fisher Scientiﬁc, MA,
45

June 2023

Medical Mass Spectrometry Vol. 7 No. 1(a): Full scan direct nanoESI mass spectra of standard samples with NaCl (middle spectrum) and KCl (lower spectrum) and control (without salt) samples (upper spectrum). (b): The expanded mass spectra of (a), amino acids (Phe,Tyr, and Trp), and caﬀeine.

Fig. 1.

The protonated ion peak of each standard is indicated by the arrowhead. The intensity of each ion peak is shown on the upper rightof each spectrum.

Fig. 2.

Averaged peak intensities of compounds in each salt spiked sample.

(a)‒(d) are detected by the positive ion mode. (e)‒(h) are detected by negative ion mode.

the analyte ions in various salt solutions detected in the pos-tendency.
＋
Na -adduct peaks were detected for both phenylalanine

itive ion mode. All the ion peaks were signiﬁcantly reducedin the salt-spiked samples (NaCl, KCl, and LiCl) comparedwith those in the samples without salt. This result shows

and tryptophan in the salt-spiked and control samples. In＋＋
the LiCl- and KCl-spiked samples, Li and K adduct ion

that high concentrations of metal ions other than sodium

peaks were not detected for phenylalanine and tryptophan,ions in the sample have similar ionization suppression

but Na adduct ion peaks were still observed. Thus, these

＋

＋

21)

＋

eﬀects . The ion suppression eﬀects decreased in the order

Na adduct ion peaks may be the result of Na eluted from

Li ＞Na ＞K , suggesting an association with ionization

the borosilicate glass surfaces of the solvent stock bottles

＋

＋

＋

46

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

and the nanoESI glass capillaries. In the NaCl-spiked sam-reason why ion suppression eﬀects were decreased indiluted single-cell sample.

＋

ples, the intensities of the Na adduct ion peaks of theseamino acids decreased, similar to the above-described behav-Comparison of ion suppression ratios with nanoESI
and conventional ESI

ior for caﬀeine. These results show that the addition of a highsalt concentration eﬀectively suppresses positive ESI.
In the negative ion detection mode, compounds such as

Next, the inﬂuence or the size of the nanoESI tip top andamino acids and citric acid were detected as deprotonatedion peaks (Fig. 2(e)‒(h)). Almost no analyte ion peaks were

the ionization suppression eﬀect of salt were investigated.

detected in samples spiked with sodium chloride or sodium

Equation 1 was used to calculate the extent by which thedihydrogen phosphate.

addition of salt reduced the peak intensity as compared toTo evaluate the ion suppression eﬀect in each sample,

the unsalted control sample.

These results show that the ion suppression eﬀect of highsalt concentrations was more signiﬁcant in the negative ion
mode than in the positive ion mode, regardless of chloride
or phosphate ions. Thus, when using MS to analyze biologi-

Relative intensity (%)＝

cal samples containing high concentrations of saline, thedetection sensitivity is signiﬁcantly lowered by the ion sup-Intensity ( Salt - spiked sample )Intensity ( Control )

×100
(1)

Fig. 3 shows the peak suppression ratio of each compound (250 nM) for each ESI method with SRM positive

pression eﬀect of ESI.
On the other hand, for all the ESI methods, the peakion detection by QQQ mass spectrometer. Although ion

intensities were improved by 10 times dilution of the salt

suppression eﬀects were also observed for the nanoESI
results in the presence of various salts, the suppressed rela-concentration. Table 2 shows the limit of detection (LOD)

tive intensity ratios were bigger than those for conventional
ESI. The ion suppression eﬀect for each ESI method is con-values for analytes in the presence and absence of NaCl.
Compared with the control samples, the samples spiked
with high concentrations of NaCl showed a 5‒25-folddecreased in sensitivity for most analytes. In contrast, whensidered to be related to the inner diameter of the capillary
used during ionization. The inner diameters of the conven-

the salt concentration was diluted 10 times, the sensitivity
only showed a 2.5-fold decrease. Thus, the salt concentra-

tional ESI capillary and the nanoESI tips were 127 μm, 3 μm,
and 10 μm, respectively. In the high-concentration NaCltion in the sample greatly aﬀects ion suppression.
For our single-cell MS, sampling volumes from sin-

samples, the peak intensities of most analytes were 20% or

gle-cells range from several hundreds of fL to a few tens ofsuppression eﬀect. Nevertheless, nanoESI with the 3 μm i.d.
tip provided improved peak intensities as compared to con-less of those in the control samples, indicating a large ion

pL. Then, 2 μL of ionized solvent is added to the nanoESI
tip to extract cellular components. Therefore, diluted sin-

ventional ESI and nanoESI with the 10 μm i.d. tip. In particular, in the 10 times diluted NaCl samples, the peak sup-gle-cell samples are expected to be less aﬀected by the ion
suppression eﬀect. In the case of single-cell MS, the sam-pression ratios with the 3 μm i.d. tip were 2‒3 times higher

pling volume from a cell is between 100 fL to 20 pL. Then,

than those with conventional ESI. As a result, the peak

2 μL of ionization solvent is added to the nanoESI tip. The

intensities were recovered to approximately 60% of thosefor the samples without NaCl. In the case of the 10 μm i.d.

Table 2. The LOD of nanoESI-MS analysis using CT-1
nanospray tip for each standard compound inhigh and low NaCl spiked samples

tip, the peak suppression ratios were similar to those forconventional ESI. These results indicate that the 3 μm i.d.
tip, which is the nanospray tip with the smallest inner diam-a

LOD (nmol/L)
H2O

NaCl

eter, minimizes the eﬀect of ion suppression by NaCl.
1/10 NaCl

Next, the relationship between these inner diameters andthe sample ﬂow rate was investigated. For nanoESI using
the 3 μm and 10 μm i.d. tips, oﬀ-line infusion with no backPhenylalanine

10

250

25

Tyrosine

100

500

250

Tryptophan

100

500

250

Caﬀeine

10

250

25

pressure was used, and sample solution spraying and ionization were automatically performed by applying a volt-: The peaks above s/n＞3.0 were used for LOD calculation.

age. The ﬂow rate of each emitter was determined by measur-a47

June 2023

Fig. 3.

Medical Mass Spectrometry Vol. 7 No. 1

Suppression rate of each substance ion peak (a): phenylalanine, (b): tyrosine, (c): tryptophan, and (d): caﬀeine
high-concentration NaCl (15.4 mM) and 10-fold diluted NaCl-spiked samples.
The bar graphs colored white show the results of ionization with conventional ESI. Similarly, the gray and black bars indicate theCT-10 and CT-1 nanospray tip, respectively.

Fig. 4.

Averaged peak intensities of compounds (Phenylalanine (a), Tyrosine (b), Tryptophan (c), and Caﬀeine (d)) which
13
were normalized with internal standard ( C labeled I.S.) in each salt spiked sample (0.9% each).

(a)‒(d) are detected by the positive ion mode. (e)‒(h) are detected by negative ion mode.
13
Averaged peak intensities which were normalized with each C labeled substance. Normalized ion peaks of each substance in highsalt concentration samples (15.4 mM each).

48

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

detected by the mass spectrometer. The ﬂow rates of the 3 μm

resulting from the exclusion-enrichment eﬀect (EEE),
which releases protons from the silanols on the inner sur-and 10 μm i.d. emitters were 68±9.5. and 180±57 nL/min,face of the nanospray tip .

ing the time at which no further ions from the sample were21)

respectively. Compared with conventional ESI (ﬂow rate:

Normalization by internal standards

100 μL/min), nanoESI is quite slow.
These results demonstrate that the inﬂuence of ion sup-To account for ﬂuctuations and the reduction in peakintensity caused by ion suppression, stable isotope-labeledpression can be reduced, even in samples with high salt
concentrations, by using a capillary with a small tip diameter and a low sample ﬂow rate during ionization. Previous

substances ( C-labeled phenylalanine, tryptophan, tyrosine,and caﬀeine) were added to the samples as internal stan-studies have reported that lower sample ﬂow rates anddards. The stable isotope dilution method, in which a com-

smaller tip inner diameters reduce the size of the charged

pound labeled with a stable isotope ( C, H,

13

13

22,23). As the rate of2

15

N, etc.) is

droplet evaporation increases with decreasing droplet size,used as an internal standard, is a useful normalization
24,25). The ionizamethod for quantitative analysis using MS

smaller charged droplets can easily reach the Rayleightion eﬃciencies of thelimit. In addition, these initial droplets are expected to havein this study were the same as those of the unlabeled forms

a low salt concentration owing to an abundance of protons

with both nanoESI and conventional ESI.

droplets in the nanoESI spraying process

Fig. 5.

13

C-labeled internal standards used

The full scan mass spectrum of a single HepG2 cytoplasm (a). The microscope capture of HepG2 cytoplasm sampling(b). Each amino acid mass spectrum (c) which is expanded from (a).
The peaks indicated by arrows are protonated amino acid ions.

49June 2023

Table 3.

Medical Mass Spectrometry Vol. 7 No. 1

The peaks of amino acid ions detected in a single HepG2 cytoplasm sample (n＝6), their average intensity, standard
deviation, and CV values, and the CV values after normalization with internal standard peaks

Amino acid
Leucine/Isoleucine

m/z
132.1022

Averaged intensity
66745

SD

CV (%)

32315

48

Normalized CV (%)

IS peak
C-Leu
C-Phe

32

13

13

Phenylalanine

166.0866

31203

16858

54

29

Tyrosine

182.0815

17835

10495

59

37

13

Tryptophan

205.0976

2833

2674

94

81

13

Serine106.0501

5565

2724

49

26

13

Threonine

120.0656

10721

5933

55

34

13

Asparagine

133.061

169

110

65

40

13

Glutamate148.0607

5045

2262

45

35

13

Proline116.0708

27877

5756

21

26

13

Valine118.0865

37701

24016

64

67

13

Lysine147.1131

21359

28857

135

114

13

Methionine

150.0586

4840

4029

83

71

13

Histidine

156.0771

5579

9171

164

150

13

Arginine

175.1193

26076

31813

122

104

13

C-Tyr
C-Trp

C-Phe
C-Phe
C-Phe
C-Phe
C-Phe
C-Phe
C-Phe
C-Phe
C-Phe
C-Phe

using stable isotope-labeled algal cell extracts as multiple

Fig. 4 shows the normalized results for the ion peak ofeach analyte in control sample and salt-spiked (NaCl, KCl,

24)

internal standards

could be applied.

and LiCl) samples. In the salt-spiked samples, the peakApplication for Single-cell analysis

intensities of most analytes decreased signiﬁcantly, as
shown in Fig. 2. However, after normalization by the corresponding stable isotope-labeled internal standards, the peak

Next, this method was applied to single-cell MS. The
cytoplasm of human hepatoma cells (HepG2 cells) was cap-

intensities were improved to almost the same level as those

tured into a nanospray tip under microscopic observation.

in the unsalted control samples. Moreover, the ﬂuctuations

In this study, we used a 3 μm i.d. nanospray tip which is ain the peak intensities were also improved. These results
indicate that normalization with the stable isotope-labeledsuitable sampling of intracellular cytoplasm components.
Then, 3 μL of solution extracted with 80% methanol con-ion peak for each analyte adequately corrects for ion suppression in the salt-containing samples and peak intensity

taining 0.1% formic acid and metabolomic QC kit solution
was added to the nanospray tip for nanoESI-Orbitrap MS

ﬂuctuations between samples.

analysis in positive ion detection mode.
Fig. 5 shows the single-cell full scan (Fig. 5(a)) andFurthermore, we investigated whether this normalization
method could be applied using a stable isotope-labeled

focused mass spectra to each amino acid ion peak (Fig.
5(c)) from single HepG2 cytoplasm sample. The micro-

internal standard and analyte with diﬀerent structures. Normalization of the caﬀeine ion peaks spiked using the ion
13peaks of C-labeled tyrosine provided no improvement in

scope capture image of a HepG2 cell sampling by nano-peak intensity, as shown in Fig. 4(d). However, the sample-to-sample ﬂuctuation (CV values) of the caﬀeine ion

detected peaks were very small due to a single cell sample,

peak improved from 15‒55% to 8.6‒26% (data not shown).

values for amino acids within a mass error of ＋/− 5 ppm.

In this case, it was diﬃcult to completely normalize the ion
suppression and ionization eﬃciency because of the diﬀer-

These peaks were not detected in the blank samples inences in the physical properties of the amino acid internal
standard and the analyte. However, as this normalization

the results, these peaks were annotated amino acids derived
from single-cell.

procedure improved peak ﬂuctuation, it could to be useful

Table 3 shows the ion peaks of the amino acids detected

for comparative analyses of samples. Furthermore, if ain the cytoplasm of a single HepG2 cell (n＝6) as well as

more detailed analysis of multiple metabolites is required,

their average intensities, standard deviations, coeﬃcient ofour previously developed universal normalization method

variation (CV) values, and CV values after normalization

spray tip is shown in Fig. 5(b). Although intensities of thethe m/z values of these peaks were close to the theoreticalwhich the cell culture medium (PBS) were sampled. From

50

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

by internal standards. The Leu, Phe, and Tyr ion peaks wereTodoroki supervised this work. The manuscript was written

normalized using the corresponding stable isotope ion

by H. Mizuno in collaboration with the other authors. All

peaks in the metabolomics QC kit, and the CV values wereauthors have approved the ﬁnal version of the manuscript.

signiﬁcantly improved. The ion peaks of the other aminoacids were normalized by the stable isotope-labeled

Acknowledgements
The authors are grateful to Dr. Kentaro Takahara, Dr.

Phe peak, which also improved the CV values. However,
the eﬀect of normalization for low-intensity ion peaks

Daisuke Higo, and Mr. Masanori Fukushima (Thermo
Fisher Scientiﬁc K.K., Japan) for performing single-cell(＜5,000) such as Asn, Trp, and Met was small, due toamino acids showed high CV values despite having highMS analysis. This study was supported in part by a Grantin-Aid for Scientiﬁc Research (19K07028) from the Minis-peak intensities. Since essential amino acids are consumed

try of Education, Culture, Sports, Science, and Technology

in large amounts during cell proliferation of cancer cellsof Japan (H.M.).

the detection sensitivity of the mass spectrometer. Somesuch as HepG2, intracellular amino acid expression levels

Conﬂicts of Interest

25)

vary depending on cell conditions . On the other hand,
Arginine is a non-essential amino acid, but it is rapidly

There are no conﬂicts to declare.

metabolized to urea and ornithine in the urea cycle. In particular, since cancer cells proliferate rapidly, it is thought that

References

the amount of arginine in the cytoplasm varies depending on

1) Mizuno H, Tsuyama N, Harada T, Masujima T: Live single-cell video-mass spectrometry for cellular and subcellu-

26)

the cells .

lar molecular detection and cell classiﬁcation. J Mass

Conclusion

Spectrom 43: 1692‒1700, 2008.

In this study, the matrix eﬀects due to salt and intracellular components in single-cell mass spectrometry using

2) Date S, Mizuno H, Tsuyama N, Harada T, Masujima T:

direct nanoESI were examined. As a result, it was con-ic cell by video mass spectrometry. Anal Sci 28: 201‒203,

ﬁrmed that the eﬀect of salt on nanoESI ionization can besuppressed by reducing the tip diameter of the nanoelectro-

3) Fukano Y, Tsuyama N, Mizuno H, Date S, Takano M, etDirect drug metabolism monitoring in a live single hepat2012.

spray tip and diluting the sample. Furthermore, by using thestable isotope-labeled metabolites of amino acids as theal: Drug metabolite heterogeneity in cultured single cells

internal standards, good normalization of amino acids andmedicine 7: 1365‒1374, 2012.

proﬁled by pico-trapping direct mass spectrometry. Nano-other metabolites was achieved. These methods wereapplied to single-cell MS to analyze amino acids in the4) Yahata K, Mizuno H, Sugiyama E, Todoroki K: Analysis

cytoplasm of HepG2 cells, and ﬂuctuations in amino acidsingle-cell mass spectrometry. J Pharm Biomed Anal 205:of the intracellular localization of amiodarone using live

peaks due to matrix eﬀects were suppressed. From this, it

114318, 2021.

was conﬁrmed that quantitative analysis of metabolites that

5) Lorenzo Tejedor M, Mizuno H, Tsuyama N, Harada T,

detect high peak intensities is possible by suppressing theinﬂuence of matrix eﬀects by direct nanoESI mass spec-

Masujima T: In situ molecular analysis of plant tissues bylive single-cell mass spectrometry. Anal Chem. 84: 5221‒

trometry.

5228, 2012.

In the future, we aim to develop this method that can6) Fujii T, Matsuda S, Lorenzo Tejedor M, Esaki T, Sakane

detect various intracellular metabolites with more sensitive

I, et al: Direct metabolomics for plant cells by live sin-and elucidate cellular phenomena at the molecular level.

gle-cell mass spectrometry. Nat Protoc 10: 1445‒1456,
2015.

Author Contributions

7) Yamamoto K, Takahashi K, Mizuno H, Anegawa A, Ishi-

Y. Kato and H. Mizuno performed the experiments andzaki K, et al: Cell-speciﬁc localization of alkaloids inanalyzed the data. H. Mizuno, S. Kudoh, and N. Tsuyama

Catharanthus roseus stem tissue measured with imaging

contributed to experimental design. E. Sugiyama and I.

MS and single-cell MS. Proc Natl Acad Sci USA 113:

Sakane contributed to analyzing data. T. Toyoʼoka and K.

113, 3891‒3896, 2016.
51

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

ces for 33 pharmaceuticals by post-column infusion. J

8) Shimizu T, Miyakawa S, Esaki T, Mizuno H, Masujima T,
et al: Live single-cell plant hormone analysis by vid-

Chromatogr B 1000: 84‒94, 2015.

eo-mass spectrometry. Plant Cell Physiol 56: 1287‒1296,

18) Stahnke H, Kittlaus S, Kempe G, Hemmerling C, Alder
L: The inﬂuence of electrospray ion source design on ma-

2015.

trix eﬀects. J Mass Spectrom 47: 875‒884, 2012.

9) Pöhö P, Lipponen K, Bespalov MM, Sikanen T, Kotiaho
T, et al: Comparison of liquid chromatography-mass spec-

19) Iavarone AT, Udekwu OA, Williams ER: Buﬀer loadingtrometry and direct infusion microchip electrospray ion-for counteracting metal salt-induced signal suppression in

ization mass spectrometry in global metabolomics of cellelectrospray ionization. Anal Chem 76: 3944‒3950, 2004.

samples. Eur J Pharm Sci 138, 104991, 2019.

20) Metwally H, McAllister RG, Konermann L: Exploring10) Hsu F-F: Mass spectrometry-based shotgun lipidomics̶

the mechanism of salt-induced signal suppression in pro-

A critical review from the technical point of view. Analtein electrospray mass spectrometry using experiments

Bioanal Chem 410: 6387‒6409, 2018.

and molecular dynamics simulations. Anal Chem 87:

11) Juraschek R, Dülcks T, Karas M: Nanoelectrospray̶

2434‒2442, 2015.

More than just a minimized-ﬂow electrospray ionization21) Hu J, Guan Q-Y, Wang J, Jiang X-X, Wu Z-Q, et al: Eﬀect

source. J Am Soc Mass Spectrom 10: 300‒308, 1999.

of nanoemitters on suppressing the formation of metal ad-12) Gethings LA, Richardson K, Wildgoose J, Lennon S, Jar-

duct ions in electrospray ionization mass spectrometry.
Anal Chem 89: 1838‒1845, 2017.

vis S, et al: Lipid proﬁling of complex biological mixturesby liquid chromatography/mass spectrometry using a nov-

22) Schmidt A, Karas M, Dülcks T: Eﬀect of diﬀerent solu-

el scanning quadrupole data-independent acquisition strat-tion ﬂow rates on analyte ion signals in nano-ESI MS, or:egy. Rapid Commun Mass Spectrom 31: 1599‒1606,

When does ESI turn into nano-ESI? J. Am Soc Mass Spec-2017.

trom 14: 492‒500, 2003.
23) Wilm M, Mann M: Analytical properties of the nanoelec-13) Gao F, McDaniel J, Chen EY, Rockwell HE, Nguyen C, ettrospray ion source. Anal Chem 68: 1‒8, 1996.

al: Adapted MS/MSALL shotgun lipidomics approach foranalysis of cardiolipin molecular species. Lipids 53: 133‒

24) Mizuno H, Ueda K, Kobayashi Y, Tsuyama N, Todoroki
K, et al: The great importance of normalization of LC-MS

142, 2018.

data for highly-accurate non-targeted metabolomics.

14) Shvartsburg AA, Isaac G, Leveque N, Smith RD, Metz
TO: Separation and classiﬁcation of lipids using diﬀeren-

Biomed Chromatogr 31: e3864, 2017.
25) Weindl D, Wegner A, Jäger C, Hiller K: Isotopologue ra-tial ion mobility spectrometry. J Am Soc Mass Spectrom
22: 1146‒1155, 2011.

tio normalization for non-targeted metabolomics. J Chromatogr A 1389: 112‒119, 2015.

15) Paglia G, Kliman M, Claude E, Geromanos S, Astarita G:
Applications of ion-mobility mass spectrometry for lipid26) Nilsson A, Haanstra JR, Engqvist M, Gerding A, Bakker

analysis. Anal Bioanal Chem 407: 4995‒5007, 2015.

BM, et al: Quantitative analysis of amino acid metabo-16) Fukui S, Sugiyama E, Mizuno H, Sakane I, Asakawa D,

lism in liver cancer links glutamate excretion to nucleo-

et al: Rapid chiral discrimination of oncometabolite

tide synthesis. Proc Natl Acad Sci USA 117: 10294‒

dl-2-hydroxyglutaric acid using derivatization and ﬁeld

10304, 2020.

asymmetric waveform ion mobility spectrometry/mass

27) Zou S, Wang X, Liu P, Ke C, Xu S: Arginine metabolism

spectrometry. J Sep Sci 44: 3489‒3496, 2021.

and deprivation in cancer therapy. Biomed Pharmacother17) Rossmann J, Gurke R, Renner LD, Oertel R, Kirch W:

118: 109210, 2019.

Evaluation of the matrix eﬀect of diﬀerent sample matri-52