Mass Spectrometic Analysis of Phosphatidylinositol PhosphatesPavlina T. Ivanova, Stephen B. Milne, Jeffrey S. Forrester, Michelle D. Armstrong and H. Alex BrownAfCS Lipidomics Lab, Department of Pharmacology & VICB, Vanderbilt University Medical Center
Figure 2. Polyphosphoinositide Enzymatic Pathways.
Figure 1. Formation of Phosphatidylinositol Phosphates.
Introduction:
Phosphatidylinositol phosphates (polyphosphoinositides) are versatile signaling lipids involved in multiple cellular functions. They and their metabolites are important elements of cellular processes including cell motility, apoptosis and cell survival, oncogenesis, vesicle transportation and fusion, calcium mobilization and release from internal stores. They are present in low concentrations within the cell, which complicates direct measurement. Methods for phosphoinositide detection in cell extracts include receptor displacement assays, metabolic labeling and chromatographic separation of the radio-labeled products after deacylation. The development of this methodology over the previous year allows us to identify a number of PIP/PIP2 species in RAW 264.7 cells that includes the chemical identification of their fatty acyl composition. Identification of the precise location of phosphates on the inositol ring (D1 through D6) requires deacylation and an additional LC-MS procedure, which must be performed in a separate extraction protocol from that used to identify the global phospholipid content. Although requiring significant effort and resources, this capability could be optimized during the next project year if designated as a priority by the Steering Committee. Using ESI-MS we were able to resolve and identify 18 polyphosphoinositides from RAW 264.7 cells.
Introduction.
Figure 3. Polyphosphoinositide Extraction Protocol.
Figure 4. Determination of Total Phosphorus.
Figure 5A. Fragmentation Pattern for 38:4 PI.
Figure 5B. Fragmentation Pattern for 38:4 PIP.
Figure 5C. Fragmentation Pattern for 38:4 PIP2.
900 920 940 960 980 1000 1020 1040 1060 1080 1100m/z
100
0
10
20
30
40
50
60
70
80
90
50
0
5
10
15
20
25
30
35
40
45
Rel
ativ
e A
bund
ance
Basal
915.5
1021.2941.3
916.5
967.3 1045.61023.5913.3965.4943.5
1030.1
911.4 1048.5969.5 995.3917.5909.6 944.5 963.4
1004.2993.5 1070.7970.3953.6939.4 1014.5 1093.61069.3 1078.6
15 min. zymosan
1021.2
1045.51023.5
941.5
967.5943.5915.4
965.51043.4 1048.6995.4
968.5 1049.4916.5913.3 944.3 1019.3969.5 993.5 996.4917.4 939.4 1031.3911.4 951.5 1069.51050.51017.5997.5918.5 937.4 1053.6977.4 1075.3991.4
1085.4
36:2
PIP
2
36:3
PIP
34:1
PIP 36
:4 P
IP
36:1
PIP
2
38:2
PIP
38:3
PIP
38:4
PIP
38:5
PIP
34:0
PIP
2
34:2
PIP
234
:1 P
IP2
38:2
PIP
2
38:3
PIP
2
38:5
PIP
238
:4 P
IP2
36:1
PIP
36:2
PIP
Figure 6. Zymosan Treatment.When treated with 50 g/mL zymosan for 15 minutes RAW 264.7 cells showed a dramatic increase in PIP and PIP2 levels compared to basal.
Figure 7. Cell Suspension Versus Scraped Cells.
A two- to three-fold increase in polyphosphoinositides was observed in unstimulated samples from cell suspensions versus scraped cell samples obtained from RAW 264.7 macrophages.
Table 1. PIP & PIP2 Levels in RAW 264.7 Cells
Stimulated with Indicated Ligands.
Figure 8. Primary Macrophage and RAW 264.7 Phosphatidylinositol
Species Comparison.Differences in the 36:2 and 38:4 phosphatidylinositol composition were found between RAW 264.7 cells and primary macrophages.
Figure 9. Comparison of PIP and PIP2 in Primary Mouse Macrophages versus RAW
264.7 Cells.Concentrations of the 36:2 and 38:4 series PIP and PIP2 species differ when compared in primary macrophages and RAW 264.7 cells.
Table 2. PI, PIP & PIP2 Species Identified in RAW 264.7 Cells.
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
885.6
581.2283.3 419.1303.3241.1
599.5223.0 259.2 435.5305.3 603.6391.4153.396.857.4
38:4 PI20:4
18:0
LPI
-H2O
-H18
:1 L
PI-H
18:0
LPA
-H2O
-H18
:1LP
A-H
2O-H
::
140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
0
510
15
20
2530
35
4045
50
5560
65
7075
80
8590
95100
Rel
ativ
e A
bund
ance
283.3419.1
303.3
241.1
419.5
281.6
296.9223.0
259.2435.5
305.3417.5
314.7439.4331.6
255.2153.3
18:0
16:0
22:4
18:1
20:3
IP-H
20-H
IP-2
H 20-
H
IP-H
GPI
-H20
-HGPI
-2H
20-H
18:1
LPA
-H18
:0 L
PA-H
18:0
LPI
-H
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m/z
0
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10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
965.5
947.7
241.2
320.8223.2
867.6303.2 342.9
419.2 885.5170.3 792.6364.2 1048.41016.1
100 150 200 250 300 350 400 450m/z
05
1015202530
3540
45
50556065
707580
859095
100
Rel
ativ
e A
bund
ance
241.2
320.8
223.2
303.2283.3
342.9
419.2
260.1
38:4 PIP
18:0
IP-H
20-H
IP-2
H20
-H IP2-H
20-H
20:4
/ IP 2
-2H
20-H
18:0
LPA
-H2O
-H
M-H
2O-H
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z
05
10
15202530354045505560
65707580
85
9095
100
Rel
ativ
e A
bund
ance
401.0
302.8
320.8
158.7222.8
176.5339.0283.0
382.8241.179.2
97.1 152.9
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5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1045.6
947.5
401.0
1027.4302.8
320.8 965.6929.5643.3158.7 723.5 785.4222.8 419.2339.0283.079.2 1009.5680.8 1067.8456.897.1 641.4
38:4PIP2
18:0
20:4
/ IP 2
-2H 2
0-H
HP2O
6-
H3P
2O7-
PO3-
H 2PO
4-
IP-H
20-H
IP-2
H20
-H
IP3-H
20-H
IP3-
2H20
-H
IP2-H
IP3-H
IP2-H
20-H
IP-3
H 20-
H
M-H
2O-H
M-H
PO3
M-H
3PO
4
http://www.signaling-gateway.org
Acknowledgments.The authors would like to thank the AfCS Cell Preparation Lab in Dallas, in particular Dianne Decamp. In addition, thanks to Andrew Goodman for excellent technical assistance.
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60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
913.4
911.3
915.6
909.6 941.41030.2
1021.5
916.5 967.5 1048.4943.3
899.6969.6
1093.7917.5 1072.3995.7945.5 1055.61043.71003.5986.6963.7 1079.0940.5 970.6 1004.4
958.4930.41012.6974.4
1021.2
1023.5
943.4
1030.2915.3 1047.3967.3
913.4 1045.2965.4
944.5916.4911.4 969.6 1048.41031.3995.5
1049.4963.2909.5 917.4 1052.2970.8955.4938.5 1069.5993.6 996.2 1019.4
919.5 1092.0979.5 1066.6928.3 1004.4
Cell suspension
Scraped
36:2
PIP
2
36:3
PIP
34:1
PIP
36:4
PIP
36:1
PIP
2
38:2
PIP
38:4
PIP
38:5
PIP
34:0
PIP
2
34:2
PIP
234
:1 P
IP2
38:2
PIP
2
38:5
PIP
238
:4 P
IP2
36:1
PIP
38:3
PIP
2
36:2
PIP
38:3
PIP