LC-MS sample preparation: Samples were prepared from 50 mL

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LC-MS sample preparation:
Samples were prepared from 50 mL cultures of exponentially growing cells cultured in either
the presence or absence of 30 mM fructose under constant illumination. The cells were
harvest by centrifugation and the resulting pellet extracted with 5 mL of HPLC grade
methanol by mixing and then incubated at -80 °C for two hours or overnight. The cell debris
was removed by centrifugation and the supernatant transferred to a fresh 50 mL tube. The cell
debris was extracted with a further 5 mL of methanol, the supernatant from which was
collected and combined with the first fraction. The cell debris were collected into a tared 1.5
mL tube, dried and the dry weight recorded. The combined methanol extractions were then
dried under a constant stream of air and the resulting residue dissolved in 3 mL dH2O. A
Waters C18 SepPak column was equilibrated with 5 mL H2O, 5 mL 100% methanol and 5 mL
H2O before the sample was passed through the column and collected in a fresh tube. The
column was then washed with a further 2 mL of H2O and combined with the first fraction.
The sample was then freeze dried and stored at -80 °C.
Mass spectrometry
The mass spectrometry of standard solutions and biological extracts was performed by
negative ion electrospray LC/MS on a Thermo Scientific LTQ XL ion trap instrument
(Thermo Fisher Scientific Inc. Waltham, MA, USA). Sedoheptulose-7-phosphate was
purchased from sigma and dissolved to a concentration of 1 mg/ml in H2O. The standard and
biological extracts were diluted in 50% acetonitrile. The freeze dried biological extracts were
re-suspended in 50% methanol (Fisher, HPLC grade) to 1 mg/ml based on the dry weight and
diluted 1 in 10 with 50% acetonitrile.
Instrument control, data acquisition, and analysis were performed using Xcalibur software
Version 2.0.7 (Thermo Fisher Scientific Inc. Waltham, MA, USA). Instrument settings were
as follows: capillary voltage −16 V, capillary temperature 350 °C, I spray voltage 3.7 kV.
High purity nitrogen was used as sheath gas at a flow rate of 60 arb. Full MS scans were
collected over the mass range m/z 160 – 500. The molecular ion m/z 289 of sedoheptulose 7phosphate (MW 290.162, Sed-7P, Sigma) was fragmented at Normalized Collision Energy 28
with isolation width 3, and the full MS spectra of sedoheptulose-7-phosphate were collected
over the mass range m/z 90 – 300 (Fig.1). sedoheptulose-7-phosphate MS spectra were
optimised using a direct infusion of a standard solution to the mass spectrometer. LC-MS was
performed on the standard diluted to 1000 ng/ml, 100 ng/ml and 10 ng/ml.
HPLC separation method
The separation system was an Accela (Thermo Fisher Scientific Inc. Waltham, MA, USA)
LC system containing a quaternary high pressure LC pump and an autosampler. The pump
system was programmed to a gradient elution as follows:
Time, min
Water, %
Acetonitrile, %
0.0
90
10
0.2
90
10
7.0
50
50
10.0
50
50
10.5
90
10
15.0
90
10
The LC column was a 1.9-µm particle size Thermo Scientific HypersilGold 150 mm x 2.1
mm i.d.10 µl of sample was injected in no waste injection mode.
maintained at 4 °C in the autosampler tray.
The samples were
E:\LTQ
E:\LTQData\ES\7743_130325123712
Data\ES\7743_130325123712
HypersilGold 150x2.1mm
u u
HypersilGold
150x2.1mm1.91.9
2512:37:12
Mar 13 12:37:12
25 Mar 13
1000inng/ml
sep-7P
1000 ng/ml sep-7P
50% ACN
22/3in 50% ACN 22/3
RT:0.00
0.00 -- 15.00
15.00 SM:
RT:
SM:13G
13G
RT:
RT:1.34
1.34
AA:
529230
AA: 529230
BP: 289.10
BP: 289.10
100
100
1
50
100
MS ICIS 7743_130325123712
50
50
0
NL: 2.85E3
m/z= 197.00-201.00 F: ITMS - c ESI Full
NL: 2.85E3
ms2 [email protected] [90.00-300.00]
m/z= 197.00-201.00 F: ITMS - c ESI Full
MS ICIS 7743_130325123712
RT: 1.33
AA: 46060
RT: 1.33
BP: 199.00
0
100
3
NL: 2.72E3
m/z= 96.00-99.00 F: ITMSNL:
- c 2.72E3
ESI Full
ms2 [email protected] [90.00-300.00]
m/z= 96.00-99.00 F: ITMS - c ESI Full
MS ICIS 7743_130325123712
ms2 [email protected] [90.00-300.00]
BP: 96.98
100
AA: 46060
BP: 199.00
100
50
ms2 [email protected] [90.00-300.00]
MS ICIS 7743_130325123712
500
0
1
2
3
4
5
6
0
Relative Abundance
Relative Abundance
0
A
7743_130325123712
RT: 12.83
AA: 139045
BP: 288.98
RT: 1.33
RT:44298
1.33
AA:
AA:96.98
44298
BP:
0
2
RT: 12.83
AA: 139045
BP: 288.98
50
0
NL: 3.87E4
NL: 3.87E4
m/z= 288.00-290.00 F: ITMS - c ESI Full
m/z= 288.00-290.00 F: ITMS - c ESI Full
ms [160.00-500.00] MS ICIS
ms [160.00-500.00] MS ICIS
7743_130325123712
1
2
3
4
5
7
8
Time (min)
6
9
10
7
8
Time (min)
11
9
12
10
13
11
14
12
15
13
14
175.09
100
NL: 5.00E4
7743_130325123712#167
RT: 1.33 AV: 1 F: ITMS
NL: 5.00E4
- c ESI Full ms
7743_130325123712#167
[160.00-500.00]
289.14
80
175.09
100
289.14
60
15
RT: 1.33 AV: 1 F: ITMS
- c ESI Full ms
[160.00-500.00]
80
40
198.16
60
163.28
20
261.03
283.17
219.08
40
0
100
20
B
163.28
97.10
198.16
199.08
219.08
80
0
100
60 97.10
339.02 357.00 372.84
311.07
261.03
283.17
424.81 438.66
480.49
NL: 3.04E3
7743_130325123712#170
424.81 438.66
480.49
RT: 1.34 AV: 1 F: ITMS
- c ESI Full ms2
NL: 3.04E3
[email protected]
7743_130325123712#170
[90.00-300.00]
339.02 357.00 372.84
311.07
199.08
RT: 1.34 AV: 1 F: ITMS
- c ESI Full ms2
[email protected]
[90.00-300.00]
40
80
20
60
400
20
229.10 244.98
271.07
169.07 191.10
113.15 139.19
100
150
297.81
200
250
169.07 191.10
113.15 139.19
300
150
200
400
450
500
297.81
0
100
350
229.10 244.98m/z
271.07
250
300
m/z
350
400
450
500
Figure 1. Mass chromatograms (1-3) and Full scan mass spectrometry of sedoheptulose-7-phosphate standard (1000 ng/ml). MS (A) was taken
from retention time 1.33 from mass chromatogram 2 and fragmentation of the molecular ion m/z 289 of sedoheptulose 7-phosphate at
Normalized Collision Energy 28 is shown in (B).
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