Page 19 - Shimadzu Journal vol.5 Issue1

Bioanalysis

1-phosphate, glucose 6-phosphate, glycerol 3-phosphate, and
Table 1 UHPLC and LC-MS/MS conditions
sedoheptulose 7-phosphate (Fig. 2). All data is shown in relative
amount compared to each metabolite amount at day 0. These results
UPLC conditions (Nexera X2 system)
indicated that amino acids, including arginine, citrulline, cysteine,
Column Discovery HS F5-3 (2.1 mm I.D. × 150 mmL, 3 µm), Sigma-Aldrich
histamine, and lysine, showed over twice amount compared to day 0
Mobile phase A 0.1% Formic acid in water
during cultivation. Mobile phase B 0.1% Formic acid in Acetonitrile
Flow rate 0.25 mL/min
Time-lapse correlation and Pathpod mapping Time program B conc. 0% (0-2.0 min) - 25% (5.0 min) - 35% (11.0 min)
- 95% (15.0 min) - 95% (20.0 min) - 0% (20.1min) - 0% (35.0 min)
According to cultivation results (Fig. 1 C), two proliferation phases Column temp. 40°C
were observed, including phase I (day 0 to 2, growth phase) and
phase II (day 3 to 5, non-growth phase). Correlativity of time-lapse MS conditions (LCMS-8050)
patterns in each phase of each metabolite in Fig. 2 were calculated Ionization ESI
and shown by Pathpod mapping (Fig. 3). Our results indicated that Ion source temp. Desolvation line: 250°C Heater block: 400°C Interface : 300°C
most metabolites in CMPs showed positive correlations (red), while Gases Nebulization: 3.0 L/min Drying : 10.0 L/min Heating : 10.0 L/min
most amino acids showed negative correlations (blue) between each MRM Transitions See Table 2
Pause time 3 msec
proliferation phases, except asparagine, aspartate, and glutamine.

A B C
25
Cell number
20
15
10 6
10
5

0
day0 day1 day2 day3 day4 day5

Fig. 1 CHO-K1 cell cultivation
CHO-K1 cell appearances were observed at day 0 (A) and day 2
(100% confluence, B). Cells were counted every 24-hour (n×3), with
cell number showed by the vertical axis (×10 ) (C). Scale= 200 µm
6

7
day0 day1 day2
6
day3 day4 day5
5

4

3
2

1

0

Fig. 2 LC-MS/MS results at day 0, 1, 2, 3, 4, and 5
LC-MS/MS results were shown in average relative value compares to pre-culture
(day 0), as the results of each metabolite at day 0 were set to 1 (n×3). Data of day
0, 1, 2, 3, 4, and 5 were shown by each colors listed in the box, respectively.

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