In Vivo Real-Time Monitoring System Using Probe ... - ACS Publications

Subscriber access provided by - Access paid by the | UCSB Libraries

In vivo real-time monitoring system using probe electrospray ionization/ tandem mass spectrometry (PESI/MS/MS) for metabolites in mouse brain Kei Zaitsu, Yumi Hayashi, Tasuku Murata, Kazumi Yokota, Tomomi Ohara, Maiko Kusano, Hitoshi Tsuchihashi, Tetsuya Ishikawa, Akira Ishii, Koretsugu Ogata, and Hiroshi Tanihata Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05291 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Title

In vivo real-time monitoring system using probe electrospray ionization/tandem mass spectrometry (PESI/MS/MS) for metabolites in mouse brain

Kei Zaitsu*1, 2, †, Yumi Hayashi1,

3, †

, Tasuku Murata4, Kazumi Yokota4, Tomomi

Ohara1, 2, Maiko Kusano2, Hitoshi Tsuchihashi2, Tetsuya Ishikawa3, Akira Ishii2, Koretsugu Ogata4, Hiroshi Tanihata4

1

In Vivo Real-time Omics Laboratory, Institute for Advanced Research, Nagoya

University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan 2

Department of Legal Medicine & Bioethics, Nagoya University Graduate

School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan. 3

Department of Radiological and Medical Laboratory Sciences, Nagoya

University Graduate School of Medicine, 1-1-20 Daiko-Minami, Higashi-ku,

Nagoya, 461-8673, Japan 4

Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto, ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

604-8511, Japan

†

Note: Kei Zaitsu and Yumi Hayashi contributed equally to this work.

*Please address all correspondence to:

Kei Zaitsu

In Vivo Real-time Omics Laboratory, Institute for Advanced Research, Nagoya

University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan

Tel.:

+81-52-744-2118;

Fax:

+81-52-744-2121;

[email protected]

2

ACS Paragon Plus Environment

E-mail:

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract

Recent improvements in ambient ionization techniques combined with mass

spectrometry has enabled to achieve real-time monitoring of analytes of interest,

even for biogenic molecules in living animals. Here, we demonstrate a

newly-developed system for in vivo real-time monitoring of metabolites in a

living mouse brain. It consists of a semi-automated manipulation system and a

unique probe electrospray ionization unit, which uses an extremely thin solid

needle (tip dia.: 700 nm) for direct sampling and ionization, coupled to a

conventional tandem mass spectrometer. The system successfully monitored 8

cerebrum

metabolites

related

to

central

energy

metabolism

in

an

isoflurane-anesthetized mouse in real time with a 20-second interval. Moreover,

our system succeeded in capturing dynamics of energy metabolism in a mouse

administered with cannabinoid type-1 receptor agonist, which is known to

disrupt cerebrum energy metabolism. The present system now opens the door

to the next stage of cutting-edge technique in achieving in vivo real-time

monitoring.

3



Introduction

Real-time monitoring of the dynamics of endogenous molecules in organisms is

one of the most difficult challenges not only in analytical chemistry but also in

biological chemistry

1-8

. There have been some effective tools using

spectroscopy, generally with fluorescent-tagging techniques, for in vivo

molecular imaging in real time, though difficulty still remains with respect to

directly monitoring “intact molecules” with high sensitivity and temporal

resolution since it generally requires both non- or low-invasive sampling as well

as rapid detection of the target molecules. Combination use of a continuous

sampling manner such as microdialysis and mass spectrometry has been

attempted for both off-line and on-line analyses of biogenic compounds for a score of years3,5,9-15. In particular, real-time monitoring of biogenic compounds using on-line analytical techniques has been achieved5,7,15, resulting in in vitro

or in vivo metabolic profiling for cellular or microbe dynamics.

We have succeeded in developing a novel approach for in vivo real-time

monitoring of metabolites using combination use of probe electrospray

4


Page 4 of 36

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ionization (PESI), a unique ambient ionization technique, and tandem mass spectrometry (MS/MS) 6. PESI, invented by Hiraoka et al., uses an extremely

thin solid needle (tip diameter: ca. 1 µm) as sampling and ionization units

16

.

Such a thin probe needle enables direct sampling with subcellular-level

invasiveness, and combinational use of PESI and single mass spectrometry (PESI/MS) has been applied to direct analysis of target compounds17-20.

However, PESI/MS lacks selectivity because PESI does not possess

chromatographic separation property. Therefore, combinational use with a

specific detector such as MS/MS is mandatory for reliable identification of metabolites. We have actually applied PESI/MS/MS to a living mouse6, where

we preliminarily succeeded in capturing the dynamics of tricarboxylic acid

(TCA)-cycle activity in the liver. However, monitoring was only capable for a

short period of time (ca. 5 min) due to incompleteness of the manipulation

system. Our team followed with a report on PESI/MS/MS to successfully detect

intact metabolites in mouse brain without any special sample preparation

despite enriched lipids and fats in the brain, which strongly interfere with

5



ionization of target metabolites21. These results strongly suggested that

PESI/MS/MS can achieve real-time monitoring of intact metabolites in the brain

of a living mouse if we construct a dedicated system combing the appropriate

manipulation unit with PESI/MS/MS: development of an accurate manipulation

system can increase repeatability of the puncture point and minimize damage to

the tissue, which is highly expected to allow for long-duration observation.

In this article, we demonstrate an in vivo real-time monitoring system, which is

composed of a semi-automatic manipulation stage and PESI/MS/MS, and its

unique features for applying to real-time monitoring of metabolites in the brain of

a living mouse. We were able to achieve real-time monitoring of 8 metabolites in

a living mouse brain for over 1 hour at a 20-second interval, and succeeded in

capturing the dynamic changes of the endogenous metabolites.

Experimental section

Apparatus setting

Fig. 1 shows the newly developed apparatus consisting of a conventional

6


Page 6 of 36

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tandem mass spectrometer (LCMS-8040) equipped with PESI ion source

(Shimadzu, Kyoto, Japan) and the manipulation system (Shimadzu). The

manipulation system was constructed with an x-y-z axial free-movable stage

(manual control for x- and y-axes, electric control for z-axis, each spatial

resolution: 3 µm), which can be manually moved to the front of the ion source

(as shown in purple arrow in Fig. 1b), and a control device. To prevent

hypothermia in the mouse from anesthesia, a rubber heater with a temperature

controller (E5EC, Omron, Kyoto, Japan) was set on the stage surface. According to our previous report6, a special sample cup (see supporting

information Fig. S4), which is fixed directly onto the mouse’s skull as detailed

later, was used for supplying ethanol to the needle tip for enhancing ionization

efficiency. To hold the cup in place, a fixing arm is also set on the stage (Fig.

1b).

Animal experiments

Animal experiments were approved by the Animal Experiment Committee of

7



Nagoya University Graduate School of Medicine (No. 28427). Experimental flow

from surgery to instrumental analysis is shown in Fig. 2. An ICR mouse (male,

10-week) was anesthetized with isoflurane and ca. 4 mm-diameter hole was

drilled at the center of the mouse’s skull to expose the intact brain surface (Fig.

2a). To prevent bleeding during analysis in advance, the meninges of the target

region were carefully removed by a 27G needle tip. A special sample cup (see

supporting Information Fig. S4) was fixed onto the mouse’s skull with an

adhesive for biotissues (Aron Alpha, Daiichi Sankyo Company, Tokyo, Japan),

and one side of the cup was secured to the head with dental acrylic resin

(Toughron Rebase, Miki Chemical Product Co. Ltd., Kyoto, Japan). After waiting

for the mouse to be awake for a while, the mouse was quickly placed onto the

stage and fixed (Fig. 2b). The mouse was anesthetized with isoflurane and

cerebrum metabolites were monitored in real time by PESI/MS/MS with 50%

ethanol solution (including 1 mg/ml heparin) supplied to the sample cup (Fig.

2c). Thirty µl of the solution was replaced with a new one every 2 min

throughout the experiment to prevent contamination and to compensate for the

8


Page 8 of 36

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


evaporative loss of ethanol.

To confirm the validity of the system, CB1R agonist (AM-2201, Ki=1.0 nM for

CB1R) in DMSO solution was injected i.p. at 30 mg/kg 3 min after the analysis

started. To confirm whether the energy metabolic dynamics observed in the

CB1R agonist-administered mouse is caused by CB1R agonist or not, the same

volume of DMSO was injected to a control mouse in the same manner as the

CB1R agonist-administered mouse.

Analytical conditions

The parameters for mass spectrometer were as follows: desolvation line

temperature, 300°C; heat block temperature, 30°C; detection mode, selected

reaction monitoring (SRM) mode; mass resolution, unit mass setting; dwell and

pause times, 24 and 1 msec, respectively. SRM transition and collision energy

for each metabolite are shown in Table 1. All targets were ionized in negative

mode, while positive ionization mode was set for cleaning the needle tip surface.

Applied voltage for the probe was +2.5 and -2.5 kV for positive and negative

9



Page 10 of 36

ionization modes, respectively. Probe insertion position was set as follows:

anteroposterior, -2.5 mm; lateral, 1 mm from the bregma. Penetration depth of

the needle was mechanically set at 1.5 mm from the brain surface (hippocampus)22. Cycle period for the prove movement was 0.05 Hz (20 sec).

Every sampling time was 30 msec. Probe stroke speed was 300 mm/sec and

acceleration was 1.00 G.

Results and discussion

Detection principle of PESI/MS/MS

As mentioned above, PESI uses a thin probe needle, made of stainless steel

with a special silicon coating, as the sampling and ionization unit. Owing to such

a thin probe needle tip (700 nm), ionization efficiency of PESI is quite high,

comparable to that of nano-electrospray ionization

20

, where ionization of the

analytes occurs just at the probe tip.

While we claim to be analyzing the internal region of the brain in this study,

there is reasonable doubt as to whether PESI is exclusively analyzing the target

10


Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


region of interest, because the probe is repeatedly inserted into the brain

passing through other regions surrounding the target region. Results from our

previous experiments, however, provide us with considerable confidence that

PESI can exclusively detect the analytes that are adsorbed on to the surface of

the probe tip. As supportive evidence, Gong et al. have demonstrated that adsorption time of analytes is directly proportional to increased sensitivity20. In

other words, in order to detect analytes by PESI/MS/MS, certain amount of

adsorption time is mandatory. While the sampling time was set at 30 msec in

this study, the rest of the probe movement time to penetrate through the brain

tissue is incomparably faster. Local analysis on the narrow target region can

thus be confidently achieved depending on the tip diameter of the probe needle.

Indeed, we have achieved in vivo real-time analysis of a living mouse liver by PESI/MS/MS using a special sample cup with 50% ethanol solution6, and have

also demonstrated that there was no elimination of the analytes on the probe

when it repeatedly passed through the 50% solution: local analysis is thus

achievable by PESI.

11



Page 12 of 36

In vivo real-time monitoring of mouse brain

All target metabolites were successfully monitored in real time in a living mouse

brain (Figs. 3 and 4). Product ion spectra of all targeted metabolites and their

authentic standards are shown in supporting information Figure S3, and all

metabolites were able to be identified by matching their product ion spectra.

As shown in Figs. 3 and 4, glutamic acid levels immediately decreased after the

analysis

started

for

both

cannabinoid

type-1

receptor

(CB1R)

agonist-administered and control mice. For the CB1R agonist-administered

mouse, CB1R agonist AM-2201 was administered i.p. 3 min after the analysis

started. As shown in Fig. 3, glucose rapidly decreased 30 min into the analysis.

At the same time, citric/isocitric acid increased remarkably. Adenosine

diphosphate (ADP) and TCA-cycle intermediates succinic, fumaric and malic

acids maintained its level or weakly increased, while α-ketoglutaric acid level

slightly deceased. This state continued for about 30 min for these metabolites,

then returned to their initial levels. From these dynamics observations, glucose

12


Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and citric/isocitric acids seem to fluctuate coordinately. Glutamic acid level

remained low throughout the monitoring period (180 min).

In contrast, a different metabolic profile was observed in the control mouse (Fig.

4). Likewise with the CB1R agonist-administered mouse, glutamic acid level

rapidly decreased after the analysis started, and remained low throughout the

monitoring period (70 min). Over the observed period, however, fluctuations in

glucose, α-ketoglutaric acid, fumaric acid, succinic acid, malic acid and ADP

levels were almost negligible. Citric/isoctirc acid levels decreased at 20 min

after the analysis started, though their levels returned to the initial state ca. 10

min later.

As mentioned above, the present system can monitor 8 cerebral metabolites

including ADP, TCA-cycle intermediates and glutamic acid in real-time for both

CB1R-agonist administered and control mice. It is well known that ADP is

localized not in the extracellular but in the intracellular region. Therefore,

monitoring of ADP fluctuation suggests that the present system can monitor in

vivo intracellular dynamics, owing to the extremely thin probe needle (tip

13



diameter: 700 nm). In fact, Gong et al. has achieved intracellular analysis for single plant-cell by PESI/mass spectrometry20, and we have also succeeded in

capturing dynamics of the TCA-cycle activity due to pyruvic acid ingestion via the portal vein in a living mouse liver by PESI/MS/MS6. Both their and our

reports support that it is highly possible to monitor intracellular dynamics by

PESI/MS/MS, even though it is unknown whether the same cell was

continuously punctured or not.

Although anesthetic isoflurane generally inhibits glutamic acid release from the

nerve cells, isoflurane can also enhance glutamic acid release in some brain areas such as the forebrain in rodents23. In addition, brain ischemia is known to increase extracellular glutamic acid level strongly24, which is even observed in anesthetic state under isoflurane25. In this study, the mouse is likely to have

produced slight ischemia from the skull-opening surgery prior to analysis;

consequently, extracellular glutamic acid level is expected to be high. In our

results, however, glutamic acid levels rapidly decreased with isoflurane

anesthesia both in control and CB1R agonist-administered mice, and remained

14


Page 14 of 36

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


low throughout the experiments, which suggests that the system may be

monitoring the intracellular region.

It is known that CB1R agonist enhances glutamic acid release, even from the astrocytes, increasing extracellular glutamic acid levels26,27. In this study,

glutamic acid level remained low for CB1R agonist-administered mouse

throughout the experiment as mentioned above, consolidating our hypothesis:

in vivo real-time monitoring of intracellular dynamics might be achieved.

To validate this hypothesis, extracellular glutamic acid levels before and after isoflurane anesthesia were monitored using microdialysis technique28 at the

same brain region. The details of the experimental conditions are described in

supporting information file. As shown in supporting information Figure S2, the

extracellular glutamic acid levels rapidly increased after isoflurane anesthesia,

which strongly supports our hypothesis.

There is, however, no appropriate manner at present to determine whether

intracellular regions were truly monitored or not by PESI/MS/MS. To validate it,

in vitro single-cell analysis by PESI/MS/MS is currently underway.

15



Dynamics of energy metabolism in CB1R agonist-administered and control

mice

The proposed dynamics observed in the CB1R agonist-administered mouse is

illustrated in Fig. 5. CB1R agonist depresses energy metabolism due to

mitochondrial dysfunction by direct stimulation of CB1R existing in the outer membrane of the mitochondria29. CB1R agonist causes some abnormal behaviours such as seizure due to acute cerebral toxic effects30,31. In our

previous experiments, these phenotypic behaviours appeared rapidly after

administration of the agonist within the first few minutes. Thus, direct effect of

CB1R agonist must be observed immediately after agonist administration.

In the present study’s results, the gradual increase in the glucose level started

immediately after CB1R agonist administration, suggesting TCA-cycle activity

suppression. About 30 min after the analysis started, however, glycolysis was

suddenly upregulated, followed by enhancement of the TCA-cycle activity,

which may be a compensation for the low energy supply. Interestingly,

16


Page 16 of 36

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


α-ketoglutaric acid level was not linked with other TCA-cycle intermediates; this

may be due to isoflurane-induced acceleration of glutamic acid release from the

cytosol to the extracellular region because α-ketoglutaric acid is rapidly

bioconverted to glutamic acid in the cytosol.

In contrast, the same low glutamic acid state was observed in the control mouse,

though the metabolic dynamics, presumably caused by CB1R agonist, observed

in the CB1R-administered mouse was not monitored in the control mouse: this is

also supportive evidence that the present system was able to capture energy

metabolism disruption caused by CB1R agonist in real time.

Effect of needle insertion on the metabolic profile

As shown in Fig. 6, neither remarkable traumatic injury nor edema were

observed on the brain surface, especially around the probe-insertion position,

examined 3 hours after in vivo real-time monitoring. Morphological damage on

the brain by needle insertion can be assumed to be practically negligible.

Although repeated needle insertion may cause cellular damages, ultra-low

17



invasiveness was achieved due to such a thin needle, which is much superior to

a microdialysis probe.

Comparison with other related works, limitation of this study and future

perspective of the present system

As mentioned above, the Caplioli research group demonstrated that the off-line

microdialysis/mass spectrometry techniques were able to monitor extracellular changes of endogenous compounds such as neuropeptides and neurotensin9-12.

In their reports, temporal resolution was mainly set at 30-minute interval due to pooling time of dialysates10-12, while temporal resolution was set at 5-minute interval for N-acetylaspartic acid monitoring9. As far as we know, the shortest

temporal resolution was achieved by Wang et al., where temporal resolution was set at 2-second interval32, though this technique was also an off-line

analytical technique. On the other hand, on-line coupling of microdialysis and mass spectrometry were reported by other research groups1,2. Deterding et al.

used a valve-switching technique for on-line preparation of dialysates and the

18


Page 18 of 36

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


temporal resolution was 2- or 5-minute interval1. Zhou et al. reported that

80-minute observation time was achieved at 2-minute interval by on-line

derivatization

and

capillary

electrophoresis

separation2.

Wong

et

al.

demonstrated on-line monitoring of melatonin for 15 hours at 30-minute interval

by combination use of microdialysis and liquid chromatography/tandem mass spectrometry with a valve-switching technique3.

Compared to such previous techniques, our methods succeeded in relatively

higher temporal resolution (20-second interval) and long-duration observation (>

60 minutes). In particular, time lag between sampling and analysis was almost

negligible in the present system, which can prevent bias caused from

degradation of the target compounds. In addition, no tedious sample

preparation such as extraction or derivatization was required. These are

noticeable advantages in the present system, though there are also limitations

to our study. The insertion depth was mechanically limited to less than 2.5 mm

from the brain surface in the present system; the target brain region therefore is

limited at present. Since we were unable to apply the system to an awake

19



mouse, further improvement of the system will be required to achieve it. Finally,

in this study, we applied the system only to mouse brain, though it is possible to

analyze other tissues such as liver and kidney, suggesting a wide-range

applicability of the present system in the near future.

Conclusion

We developed an in vivo real-time monitoring system for mouse brain by

combinational use of PESI/MS/MS and a semi-automatic manipulation unit. To

validate the system, we applied the system to CB1R-agonist administered and

control mice brains, successfully capturing the metabolic dynamics in mouse

brain over 1 hour with 20-second intervals. The obtained results for

CB1R-agonist administered mouse were reasonable, demonstrating the

practicality of the present system. Since this system has a high potential for

application not only to other mouse tissues but also to other fields such as

plants, a new research field called “Real-time metabolomics” can be expected to

expand in the future using this system.

20


Page 20 of 36

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acknowledgements

This research was partially supported by the program for promoting the

enhancement of research universities from Ministry of Education, Culture,

Sports, Science and Technology (MEXT) in Japan.

Conflict of interest

There is no conflict of interest to declare.

Author contributions

K.Z. and Y.H. contributed equally to this work: K.Z. and Y.H. conceived the idea,

designed and supervised the experiments, and wrote the manuscript. T.M., K.Y.,

K.O. and H. Tanihata designed and fabricated the apparatus. K.Z. and Y.H.

performed the entire experiments, and T.O. partially executed the experiments.

K.Z., Y.H., T.M., K.Y., K.O. and H. Tanihata discussed the results. M.K., H.

Tsuchihashi, T.I. and A.I. commented on the manuscript.

21



Supporting Information Available: [Supporting information of In vivo real-time

monitoring system using PESIMSMS for metabolites in mouse brain]

22


Page 22 of 36

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures and table

Fig. 1 Newly-developed in vivo real-time monitoring system. (a) Photograph of

the overall system. The system consists of a free-movable stage, PESI

actuator and conventional tandem mass spectrometer. (b) Close up

photograph of the orange dotted square section in (a) and a picture of the

PESI probe. The free-movable stage can be manually moved to the front of the

mass spectrometer (purple arrow).



Fig. 2 Experimental flow from surgery to instrumental analysis. (a) Illustration of

the surgery for opening the skull on stereotaxic frame and the photograph of

the opened skull (CB1R agonist-administered mouse). (b) Mouse secured to

the fixing arm with a special sample cup fixed to the mouse’s head. (c)

Photograph of in vivo real-time monitoring.

24


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 3 In vivo real-time monitoring of the brain of a CB1R agonist-administered

mouse. Yellow arrow: CB1R agonist injection point, Light yellow zone: period in

which dynamic changes of energy metabolism was observed. Run time: 180

min.



Fig. 4 In vivo real-time monitoring of the brain of a control mouse. Yellow arrow:

injection point of vehicle (DMSO). Run time: 70 min.

26


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 5 Schematic representation of the proposed dynamics observed in CB1R

agonist-administered mouse.

27



Fig.

6

Stereomicroscope

image

of

the

Page 28 of 36

brain

agonist-administered mouse after in vivo monitoring.

28


surface

of

CB1R

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1 SRM transition and collision energy for each metabolite Compound name

SRM transition

Collision energy (V)

ADP

m/z 426 > 159

30

Citric acid/isocitric acid

m/z 191 > 111

20

Fumaric acid

m/z 115 > 71

15

Glucose

m/z 179 > 59

20

Glutamic acid

m/z 146 > 102

20

α-Ketoglutaric acid

m/z 145 > 101

10

Malic acid

m/z 133 > 115

20

Succinic acid

m/z 117 > 73

20

29



References

(1) Deterding, L. J.; Dix, K.; Burka, L. T.; Tomer, K. B. Anal. Chem. 1992, 64,

2636-2641.

(2) Zhou, S. Y.; Zuo, H.; Stobaugh, J. F.; Lunte, C. E.; Lunte, S. M. Anal. Chem.

1995, 67, 594-599.

(3) Wong, P. S. H.; Yoshioka, K.; Xie, F.; Kissinger, P. T. Rapid Commun. Mass

Spectrom. 1999, 13, 407-411.

(4) Motta, A.; Paris, D.; Melck, D. Anal. Chem. 2010, 82, 2405-2411.

(5) Hsu, C.-C.; ElNaggar, M. S.; Peng, Y.; Fang, J.; Sanchez, L. M.; Mascuch, S.

J.; Møller, K. A.; Alazzeh, E. K.; Pikula, J.; Quinn, R. A.; Zeng, Y.; Wolfe, B. E.;

Dutton, R. J.; Gerwick, L.; Zhang, L.; Liu, X.; Månsson, M.; Dorrestein, P. C.

Anal. Chem. 2013, 85, 7014-7018.

(6) Zaitsu, K.; Hayashi, Y.; Murata, T.; Ohara, T.; Nakagiri, K.; Kusano, M.; 30


Page 30 of 36

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nakajima, H.; Nakajima, T.; Ishikawa, T.; Tsuchihashi, H.; Ishii, A. Anal. Chem.

2016, 88, 3556-3561.

(7) Bavli, D.; Prill, S.; Ezra, E.; Levy, G.; Cohen, M.; Vinken, M.; Vanfleteren, J.;

Jaeger, M.; Nahmias, Y. Proceedings of the National Academy of Sciences

2016, 113, E2231-E2240.

(8) Jeong, S.; Eskandari, R.; Park, S. M.; Alvarez, J.; Tee, S. S.; Weissleder, R.;

Kharas, M. G.; Lee, H.; Keshari, K. R. Science Advances 2017, 3.

(9) Lin, S.-N.; Slopis, J. M.; Butler, I. J.; Caprioli, R. M. J. Neurosci. Methods

1995, 62, 199-205.

(10) Emmett, M. R.; Andrén, P. E.; Caprioli, R. M. J. Neurosci. Methods 1995,

62, 141-147.

(11) Andrén, P. E.; Emmett, M. R.; DaGue, B. B.; Steulet, A.-F.; Waldmeier, P.;

31



Caprioli, R. M. J. Mass Spectrom. 1998, 33, 281-287.

(12) Andrén, P. E.; Caprioli, R. M. Brain Res. 1999, 845, 123-129.

(13) Nandi, P.; Lunte, S. M. Anal. Chim. Acta 2009, 651, 1-14.

(14) Behrens, H. L.; Li, L. In Peptidomics: Methods and Protocols, Soloviev, M.,

Ed.; Humana Press: Totowa, NJ, 2010, pp 57-73.

(15) Link, H.; Fuhrer, T.; Gerosa, L.; Zamboni, N.; Sauer, U. Nature Methods

2015, 12, 1091.

(16) Hiraoka, K.; Nishidate, K.; Mori, K.; Asakawa, D.; Suzuki, S. Rapid

Commun. Mass Spectrom. 2007, 21, 3139-3144.

(17) Yoshimura, K.; Chen, L. C.; Asakawa, D.; Hiraoka, K.; Takeda, S. J. Mass

Spectrom. 2009, 44, 978-985.

(18) Mandal, M. K.; Chen, L. C.; Hashimoto, Y.; Yu, Z.; Hiraoka, K. Analytical

32


Page 32 of 36

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Methods 2010, 2, 1905-1912.

(19) Saha, S.; Mandal, M. K.; Hiraoka, K. Analytical Methods 2013, 5,

4731-4738.

(20) Gong, X.; Zhao, Y.; Cai, S.; Fu, S.; Yang, C.; Zhang, S.; Zhang, X. Anal.

Chem. 2014, 86, 3809-3816.

(21) Hayashi, Y.; Zaitsu, K.; Murata, T.; Ohara, T.; Moreau, S.; Kusano, M.;

Tanihata, H.; Tsuchihashi, H.; Ishii, A.; Ishikawa, T. Anal. Chim. Acta 2017, 983,

160-165.

(22) George Paxinos, K. F., George Paxinos, Keith Franklin. Paxinos and

Franklin's the Mouse Brain in Stereotaxic Coordinates 4th Edition; Academic

Press, 2012.

(23) Dong, M. D. P. D. H.-l.; Fukuda, M. D. P. D. S.; Murata, D. V. M. M. S. E.;

33



Higuchi, M. D. P. D. T. Anesthesiology 2006, 104, 122-133.

(24) Nishizawa, Y. Life Sci. 2001, 69, 369-381.

(25) Patel, M. D. F. P. M.; Drummond, M. D. F. J. C.; Cole, M. D. D. J.;

Goskowicz, M. D. R. L. Anesthesiology 1995, 82, 996-1003.

(26) Pistis, M.; Ferraro, L.; Pira, L.; Flore, G.; Tanganelli, S.; Gessa, G. L.;

Devoto, P. Brain Res. 2002, 948, 155-158.

(27) Ferraro, L.; Tomasini, M. C.; Gessa, G. L.; Bebe, B. W.; Tanganelli, S.;

Antonelli, T. Cereb. Cortex 2001, 11, 728-733.

(28) Fuwa, T.; Suzuki, J.; Tanaka, T.; Inomata, A.; Honda, Y.; Kodama, T. The

Journal of Toxicological Sciences 2016, 41, 329-337.

(29) Benard, G.; Massa, F.; Puente, N.; Lourenco, J.; Bellocchio, L.;

Soria-Gomez, E.; Matias, I.; Delamarre, A.; Metna-Laurent, M.; Cannich, A.;

34


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hebert-Chatelain, E.; Mulle, C.; Ortega-Gutierrez, S.; Martin-Fontecha, M.;

Klugmann, M.; Guggenhuber, S.; Lutz, B.; Gertsch, J.; Chaouloff, F.;

Lopez-Rodriguez, M. L.; Grandes, P.; Rossignol, R.; Marsicano, G. Nat.

Neurosci. 2012, 15, 558-564.

(30) Zaitsu, K.; Hayashi, Y.; Suzuki, K.; Nakayama, H.; Hattori, N.; Takahara, R.;

Kusano, M.; Tsuchihashi, H.; Ishii, A. Life Sci. 2015, 137, 49-55.

(31) Wallace, M. J.; Blair, R. E.; Falenski, K. W.; Martin, B. R.; DeLorenzo, R. J.

J. Pharmacol. Exp. Ther. 2003, 307, 129-137.

(32) Wang, M.; Slaney, T.; Mabrouk, O.; Kennedy, R. T. J. Neurosci. Methods

2010, 190, 39-48.

35



for TOC only

36


Page 36 of 36

In Vivo Real-Time Monitoring System Using Probe ... - ACS Publications

Recommend Documents