1
Supplemental information
2
Supplemental figure legends
3
4
Figure S1. Sections of cerebellum ( x 10) containing the molecular and granule layers
5
stained with hematoxylin and eosin, following (a) sacrifice by microwave irradiation at
6
5kW for 0.94 s and (b) sacrifice by decapitation. Several 'scratches', those were made
7
during cryosectioning were found in FMW-treated sections due to its fragile property.
8
However, the nucleus structures were intact and interfaces among molecular layer,
9
granule layer and white matter were clearly defined.
10
W: white matter, ML: molecular layer, GL: granule layer.
11
12
Figure S2.
13
Mass spectra obtained from the brain sections those were treated with FMW, ISF and PEF(1min).
14
The mass peaks corresponding to metabolites whose distributions were visualized in figure 2 were
15
highlighted.
16
17
Figure S3.
18
Mass spectra obtained from the brain sections prepared from non-labeled (green) and
19
administered mice (red). The mass peaks corresponding to non-labeled and 13C-labled compounds of
1
13C
6-glucose
1
glutamate and UDP-glucose were highlighted. Their brain distributions were visualized in figure 4.
2
3
Supplemental methods
4
Quantification of Metabolites by CE-ESI-MS.
5
All CE-ESI-MS experiments were performed using an Agilent CE System equipped
6
with an air pressure pump, an Agilent 6520 Accurate Q-Tof mass spectrometer, an
7
Agilent 1200 series isocratic high-performance LC pump, 7100 CE-system, a G1603A
8
Agilent CE-MS adapter kit, and a G1607A Agilent CE-MS sprayer kit (Agilent
9
Technologies). All chemical standards were dissolved in ultrapure water, 0.1 N HCl, or
10
0.1 N NaOH to obtain 10 mM or 100 mM stock solutions. Working standard solutions
11
were prepared by diluting stock solutions with ultrapure water just before injection.
12
For cationic metabolites, a fused silica capillary tube (50 μm inner diameter × 1 m total
13
length) was used for separation (1). The electrolyte for CE separation was 1 M formic
14
acid. Each new capillary was flushed successively with the running electrolyte for 20
15
min. Before each injection, the capillary was preconditioned for 5 min by flushing with
16
the running electrolyte. Approximately 3 nL of sample solution was injected at 5 kPa for
17
3.0 s, with 30 kV of voltage applied. The capillary temperature was maintained at 20
18
°C, and the sample tray was cooled to below 5 °C. Methanol/water (50% vol/vol)
19
containing 5 mM ammonium acetate was delivered as the sheath liquid at 10 μL min−1 .
2
1
ESI-MS was conducted in the positive-ion mode, and 4 kV of the ion spray voltage was
2
applied after sample injection. A flow rate of heated dry nitrogen gas (heater
3
temperature 300 °C) was maintained at 69 kPa.
4
For MS, m/z region from 100 to 1000 was measured and deprotonated [M+H]+ ions
5
were used for quantification of cationic metabolites of interest.
6
For anionic metabolites, a cationic polymer-coated COSMO (+) capillary tube (50 μm
7
inner diameter × 1 m total length; Nacalai Tesque) was used for separation (2). The
8
electrolyte used for CE separation was 50 mM ammonium acetate solution (pH 8.5).
9
Each new capillary was flushed successively with running buffer (50 mM ammonium
10
acetate solution; pH 8.5), 50 mM acetic acid (pH 3.4), and then running buffer again, for
11
20 min each. In addition, before each injection, the capillary was flushed with 50 mM
12
acetic acid (pH 3.4) for 2 min, followed by the running electrolyte for 5 min.
13
Samples were injected at 5 kPa for 30 s (∼30 nL). The applied voltage was set at −30
14
kV. The capillary temperature was maintained at 20 °C, and the sample tray was cooled
15
to below 5 °C. Methanol-water (50% vol/vol) containing 5 mM ammonium acetate was
16
delivered as the sheath liquid at 10 μL min−1 . ESI-MS was conducted in the negative-
17
ion mode, with the ion spray voltage set to 3.5 kV. A flow rate of heated dry nitrogen
18
gas (heater temperature 300 °C) was maintained at 69 kPa. For MS, m/z region from
3
1
100 to 1000 was measured and deprotonated [M-H]− ions were used for quantification
2
of anionic metabolites.
3
Quantification of metabolites was performed with MassHunter QUAL-Software
4
(Agilent Technologies). In the quantification procedure, we at first obtained mass
5
chromatograms of each standard compound by using accurate mass (less than 10 ppm)
6
and determined their unique migration time. Then, by using calibration curves those
7
were calculated from measurement results of dilution series of standard compounds,
8
absolute concentrations of the specimen metabolites were acquired.
9
10
References
11
1. Soga T, Heiger DN (2000) Amino acid analysis by capillary electrophoresis electrospray
12
ionization mass spectrometry. Anal Chem 72:1236–1241.
13
2. Soga T, et al. (2002) Simultaneous determination of anionic intermediates for Bacillus
14
subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass
15
spectrometry. Anal Chem 74:2233–2239.
16
17
18
4
1
Supplemental table 1.
2
Absolute concentrations of brain metabolites measured by CE/ESI-MS prepared by the four different
3
sampling methods.
nmol/g_weight
4
PM F (10m in)
PM F (1m in)
ISF
FM W
AV
SD
AV
SD
AV
SD
AV
SD
Glyc olysis
Gluc os e 1-phos phate
Gluc os e 6-phos phate
Fruc tos e 6-phos phate
Fruc tos e 1,6-diphos phate
Dihy drox y ac etonephos phate
2,3-Diphos phogly c erate
3-Phos phogly c erate
Phos phoenolpy ruv ate
Py ruv ate
Lac tate
20.0
14.9
7.4
61.2
0.4
11.3
32.8
5.6
13.4
7842.3
2.6
2.1
1.6
15.6
0.5
4.4
13.7
2.6
9.3
1027.3
41.3
67.2
14.7
114.2
5.3
49.1
30.6
8.1
26.1
6422.6
11.1
19.9
3.3
42.8
4.2
14.3
9.9
1.6
21.3
1010.1
30.9
37.1
17.2
267.7
2.9
134.5
83.0
17.5
30.7
3510.6
5.3
8.7
3.4
37.4
1.6
31.5
13.8
4.8
8.9
350.9
17.8
152.8
94.9
86.3
0.2
115.7
19.3
15.6
40.1
1561.7
0.8
7.5
4.2
4.5
0.4
9.1
1.1
1.4
16.1
264.7
Glyc e rol ph osph ate sh u ttle
Gly c erol 3-phos phate
730.7
273.8
438.7
63.9
232.6
31.1
158.1
6.9
TCA Cyc le
Ac ety l CoA
Citrate
c is -Ac onitate
Is oc itrate
2-Ox oglutarate
Suc c inate
Fum arate
M alate
3.3
159.8
2.8
6.7
0.0
1074.5
72.9
361.9
1.1
22.9
0.7
6.9
0.0
451.2
35.2
152.5
4.3
230.3
5.6
4.9
3.2
516.6
112.8
426.1
1.0
23.7
0.8
1.5
3.0
79.4
19.8
65.9
1.3
438.6
6.9
7.9
15.0
86.1
78.2
522.2
0.1
67.0
1.7
3.5
6.9
11.9
15.6
81.7
0.7
430.9
20.9
14.7
149.5
183.6
146.9
339.6
0.3
45.9
1.3
1.2
24.7
21.6
13.3
18.5
Pe n tose ph osph ate path way
Gluc os e 6-phos phate
6-Phos phogluc onate
Ribulos e 5-phos phate
Ribos e 5-phos phate
D-Sedoheptulos e 7-phos phate
Fruc tos e 6-phos phate
DL-Gly c eraldehy de 3-phos phate
14.9
10.4
103.4
0.0
12.5
7.4
0.4
2.1
3.2
25.0
0.0
8.4
1.6
0.9
67.2
14.1
28.4
2.2
11.9
14.7
0.0
19.9
2.3
7.1
1.0
3.1
3.3
0.0
37.1
10.2
13.8
0.0
13.3
17.2
0.0
8.7
1.4
2.7
0.0
2.4
3.4
0.0
152.8
13.0
14.0
0.2
44.1
94.9
0.0
7.5
0.7
2.7
0.4
3.2
4.2
0.0
Nu c le ic ac id syn th e sis
5-Phos phoribos y l-py rophos phate
0.6
0.4
5.4
0.9
8.0
0.9
6.6
1.3
Me th ylate d produ c ts
M et
S-Adenos y l-L-m ethionine
Sperm idine
Sperm ine
S-Adenos y l-L-hom oc y s teine
Cy s tathionine
Hom os erine
Taurine
Glutathione, reduc ed form
Glutathione, ox idiz ed form
N-M ethy l-Arg
ADM A
SDM A
34.4
24.8
15.8
1.7
3.7
25.8
4.5
7864.5
261.3
926.3
0.8
1.2
2.0
8.8
4.5
3.2
0.3
2.0
14.7
3.1
1212.6
116.1
280.6
1.2
1.2
2.8
29.6
22.3
20.0
3.0
1.2
18.6
9.5
8123.5
370.0
1363.4
0.5
0.8
0.9
5.7
2.3
8.6
1.0
0.4
3.8
5.4
1064.1
509.2
246.4
0.2
0.1
0.2
17.9
21.0
16.5
3.1
0.8
18.6
10.5
7022.9
16.1
1238.2
0.1
0.3
0.5
4.3
2.4
2.5
0.4
0.1
3.6
1.6
952.9
3.0
213.7
0.1
0.0
0.1
22.2
9.9
14.2
3.8
0.4
19.7
16.3
8848.0
32.7
1603.8
0.2
0.5
0.7
2.8
2.0
1.6
1.2
0.4
1.6
2.4
610.8
23.5
159.1
0.2
0.1
0.2
Ure a c yc le
Glu
Ornithine
Citrulline
Arg
Creatine
Creatine phos phate
Creatinine
Hy drox y proline
7558.0
18.5
132.8
86.3
2819.3
16.8
28.7
16.1
850.2
8.4
22.4
21.1
584.4
5.6
2.6
7.3
8024.9
8.6
122.3
74.5
2664.8
12.4
22.5
18.6
1219.4
0.6
18.5
12.2
333.2
4.0
2.1
5.0
6956.1
13.7
143.5
59.0
2258.7
807.3
24.1
20.6
953.5
2.4
24.7
9.4
256.7
318.0
3.8
2.3
8134.2
11.6
175.4
69.0
1454.4
2554.1
101.4
34.5
524.4
2.7
11.2
2.8
972.3
136.7
16.1
3.7
Amin o ac ids
Gly
Ala
Ser
Thr
Val
Ile
Leu
Ly s
Arg
His
Ty r
Phe
M et
Cy s
Pro
Gln
Glu
As n
As p
β-Ala
1076.6
978.4
793.6
114.8
107.7
43.9
96.4
208.7
86.3
132.3
107.1
96.3
34.4
0.0
87.1
3901.1
7558.0
106.9
2475.8
108.2
109.1
66.4
54.4
27.4
12.0
11.5
46.3
8.2
21.1
59.8
39.4
41.0
8.8
0.0
15.2
1472.8
850.2
6.0
102.6
50.3
994.8
648.8
802.3
105.2
89.7
27.3
45.7
210.8
74.5
83.3
73.9
62.5
29.6
0.0
68.8
2899.8
8024.9
91.8
2876.6
65.3
123.2
110.6
100.2
15.7
14.4
2.1
7.8
37.1
12.2
9.3
9.9
10.8
5.7
0.0
11.0
426.2
1219.4
10.4
412.8
12.4
1003.9
594.1
757.3
84.3
91.6
30.6
50.1
202.5
59.0
80.6
58.0
49.5
17.9
0.0
64.7
2773.2
6956.1
90.6
2188.1
64.5
82.3
113.3
90.4
5.4
9.6
0.4
9.3
23.9
9.4
8.9
11.5
8.4
4.3
0.0
10.3
326.9
953.5
8.3
236.2
9.3
1055.7
557.7
797.9
94.4
98.9
16.2
40.1
200.7
69.0
89.7
81.6
60.9
22.2
0.0
72.5
3008.5
8134.2
96.5
2976.2
52.5
53.5
52.9
44.4
7.1
14.3
4.6
6.9
15.6
2.8
6.4
5.8
5.9
2.8
0.0
5.1
144.1
524.4
8.3
162.6
4.6
Nu c le ic ac ids
Adenine
Guanine
Cy tos ine
Urac il
Adenos ine
Guanos ine
Cy tidine
Inos ine
AM P
GM P
CM P
TM P
IM P
ADP
GDP
CDP
TDP
ATP
GTP
CTP
UTP
dATP
Hy pox anthine
Xanthine
NAD
NADH
NADP
NADPH
FAD
13.2
0.0
1.8
83.1
242.3
33.9
40.1
293.8
2123.7
385.1
18.6
0.0
505.9
281.7
250.4
1.2
1.3
105.8
85.5
0.0
1.4
0.7
100.3
19.8
239.9
122.6
25.3
2.5
6.1
9.5
0.0
2.0
16.6
112.1
5.8
1.0
39.8
654.2
107.6
7.2
0.0
260.5
132.2
111.7
0.8
1.0
28.8
39.1
0.0
0.9
0.5
24.5
13.8
65.7
22.4
11.6
1.8
4.1
2.8
1.2
0.7
23.6
112.0
12.0
15.2
34.1
2156.0
244.8
37.9
1.1
356.2
586.2
268.2
12.2
2.4
445.2
174.5
7.3
19.3
3.4
15.0
7.9
290.7
175.3
22.6
5.5
10.4
1.7
2.4
0.3
4.1
24.7
4.9
2.7
8.4
254.5
46.6
4.2
0.9
63.7
141.3
56.8
2.8
1.6
81.9
34.2
1.5
1.8
0.5
4.3
2.5
98.7
90.3
8.2
1.2
2.3
1.4
0.0
0.5
10.5
19.7
2.3
3.3
7.7
552.8
34.1
20.2
0.6
74.7
686.1
180.8
25.0
2.4
2198.0
456.3
45.6
125.1
14.6
6.1
6.0
322.5
59.8
29.7
3.3
9.4
0.3
0.0
0.1
2.0
5.7
0.9
0.5
2.5
321.8
13.8
10.4
0.7
38.2
184.0
49.6
5.6
0.6
429.0
74.7
13.8
37.0
2.7
1.4
1.2
56.4
13.3
9.9
0.4
0.6
4.1
0.0
0.5
8.0
1.0
1.9
3.5
4.9
197.7
31.8
7.1
0.2
28.4
870.0
201.6
27.9
0.7
2780.5
573.3
96.8
209.2
18.1
5.3
3.1
327.4
36.4
8.7
32.3
12.0
0.8
0.0
0.2
1.7
0.1
1.4
1.2
0.9
42.4
3.3
1.4
0.4
4.8
115.5
48.6
2.8
0.8
519.6
66.6
8.8
24.8
2.7
1.3
3.6
68.8
7.9
1.8
4.0
1.5
Fatty ac id me tabolism
L-Carnitine
CoA
Ac ety l CoA
M alony l CoA
95.9
6.4
3.3
35.2
11.6
2.2
1.1
5.9
79.0
8.8
4.3
160.5
16.8
6.4
1.0
24.8
86.5
0.0
1.3
637.0
13.9
0.0
0.1
131.3
128.2
0.3
0.7
884.7
11.5
0.6
0.3
113.7
Tryptoph an me tabolism
Anthranilate
0.2
0.5
0.0
0.0
0.0
0.0
0.0
0.0
Cre atin e me tabolism
Creatine
Creatine phos phate
Creatinine
2819.3
16.8
28.7
584.4
5.6
2.6
2664.8
12.4
22.5
333.2
4.0
2.1
2258.7
807.3
24.1
256.7
318.0
3.8
1454.4
2554.1
101.4
972.3
136.7
16.1
Cate c h lamin e me tabolism
DOPA
Dopam ine
L-Norepinephrine
0.0
85.7
0.0
0.0
23.8
0.0
0.0
81.3
0.0
0.0
10.9
0.0
0.0
61.1
0.0
0.0
6.0
0.0
0.0
58.1
0.0
0.0
5.1
0.0
Oth e rs
Carnos ine
FAD
107.4
12.8
32.1
9.8
99.5
12.1
24.1
7.4
83.4
12.3
19.7
2.8
98.4
9.8
23.8
2.7
5
1
6