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

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50

0

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25

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35

40

45

Rel

ativ

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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

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25

30

35

40

45

50

55

60

65

70

75

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90

95

100

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bund

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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

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40

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50

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100

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bund

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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

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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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30

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45

50

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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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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