Mass Spectrometic Analysis of Phosphatidylinositol PhosphatesPavlina T. Ivanova, Stephen B. Milne, Jeffrey S. Forrester, Michelle D. Armstrong and H. Alex Brown

AfCS 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

1021.2

941.3

916.5

967.3 1045.61023.5913.3965.4943.5

1030.1

911.4 1048.5969.5

995.3917.5909.6 944.5 963.41004.2

993.5 1070.7970.3953.6939.4 1014.5 1093.61069.3 1078.6

15 min. zymosan

1021.2

1045.5

1023.5

941.5

967.5

943.5915.4

965.51043.4 1048.6995.4

968.5 1049.4916.5913.3 944.3

1019.3969.5 993.5996.4917.4 939.4 1031.3911.4 951.5 1069.51050.5

1017.5997.5918.5 937.4 1053.6977.4 1075.3991.41085.4

36:

2 P

IP2

36:

3 P

IP

34:

1 P

IP 36:

4 P

IP

36:

1 P

IP2

38:

2 P

IP3

8:3

PIP

38:

4 P

IP3

8:5

PIP

34:

0 P

IP2

34:

2 P

IP2

34:

1 P

IP2

38:

2 P

IP2

38:

3 P

IP2

38:

5 P

IP2

38:

4 P

IP2

36:

1 P

IP3

6:2

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

Re

lativ

e A

bu

nda

nce

885.6

581.2283.3 419.1303.3

241.1599.5223.0 259.2 435.5305.3 603.6391.4153.396.857.4

38:4 PI20:4

18:0

LP

I-H

2O-H

18:1

LP

I-H

18:0

LP

A-H

2O-H

18:1

LP

A-H

2O-H

::

140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

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

bu

nd

ance

283.3419.1

303.3

241.1

419.5

281.6

296.9

223.0

259.2435.5

305.3

417.5314.7

439.4331.6255.2153.3

18:0

16:0

22:4

18:1

20:3

IP-H

20-H

IP-2

H20

-H

IP-H

GP

I-H

20-HG

PI-

2H20

-H

18:1

LP

A-H

18:0

LP

A-H

18:0

LP

I-H

100 200 300 400 500 600 700 800 900 1000 1100

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

965.5

947.7

241.2

320.8

223.2867.6

303.2 342.9419.2 885.5170.3 792.6364.2 1048.41016.1

100 150 200 250 300 350 400 450m/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

bu

nd

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-

H20

-H

20:4

/ IP

2-2H

20-H

18:0

LP

A-H

2O-H

M-H

2O-H

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/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

bu

nd

ance

401.0

302.8

320.8

158.7222.8

176.5339.0283.0

382.8241.179.2

97.1 152.9

100 200 300 400 500 600 700 800 900 1000 1100m/z0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

1045.6

947.5

401.0

1027.4

302.8320.8 965.6929.5643.3158.7 723.5 785.4222.8 419.2339.0283.079.2 1009.5680.8 1067.8456.897.1 641.4

38:4PIP2

18:0

20:4

/ IP

2-2H

20-H

HP

2O6-

H3P

2O7-

PO

3-

H2P

O4-

IP-H

20-H

IP-2

H20

-H

IP3-

H20

-H

IP3-

2H20

-H

IP2-

H

IP3-

H

IP2-

H20

-H

IP-3

H20

-H

M-H

2O-H

M-H

PO

3

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.

900 920 940 960 980 1000 1020 1040 1060 1080 1100

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nda

nce

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 1055.61043.71003.5

986.6963.7 1079.0940.5 970.6 1004.4958.4930.4

1012.6974.4

1021.2

1023.5

943.4

1030.2915.3 1047.3

967.3913.4

1045.2965.4

944.5

916.4911.4969.6 1048.4

1031.3995.51049.4

963.2909.5

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