Multiplexed lipid arrays of ligand-induced changes The Alliance for Cell Signaling 2003 Meeting-...

Multiplexed lipid arrays of ligand-induced changes

The Alliance for Cell Signaling2003 Meeting- Pasadena, CA

Lipidomics LaboratoryH. Alex Brown, director

Vanderbilt University Medical Center

Glycerol-3-phosphate Dihydroxyacetone phosphate Choline

1-acyl-G-3-P 1-acyl-dhAP

PA

CDP-DG

PI

PIP

PIP2

PGp PG

Cardiolipin

LPP

DG

CPT

CDP-Choline

PC

PE

EPT

CDP-Ethanolamine

ET

Ethanolamine-P

CK/EK

Ethanolamine

Choline-P

CK/EK

CT

SM

PEMTPSD

PSS

Serine Ethanolamine

CO2

PS

Phospholipid Biosynthetic Pathways

PIP3

PI3K

PLCIP3 + DG

DGK

CLS

LPA

Lyso-bis-PA

Conduct lipid analysis on instrumentation with selective ion scanning capability and at least moderate throughput capacity (greater than 100 samples/week).

Construct a working phospholipids fragmentation library for B-lymphocytes

(subsequently refocused by Steering Cmte to WEHI-231 cells). Generate new computer program software to achieve computational

analysis of mass spectrometry data to detect qualitative changes in membrane lipid composition (cellular lipid analysis program).

Initiate LIPID ARRAYS on ligand stimulated WEHI-231 cells. Determine feasibility of measuring poly-phosphatidylinositol species as part

of the LIPID ARRAYS.

Review of Aims 2002-03 (Lipidomics Lab)

Identification of lipid species

Conduct lipid analysis on instrumentation with selective ion scanning capability and moderate throughput capacity (greater than 100

samples/week).

Electrospray Mass Spectrometry

Detector

Q1ElectrosprayIon Source

Q3Collision Cell (Q2)

Syringe Pump Tandem Mass Spectrometer

CID: Monitor product formation in Q3 from a specific parent in Q1SIM: Monitor specific m/z in Q1SRM: Monitor transition of a specific m/z in Q1 to a specific product in Q3

D. Hachey

Adaptation to triple-quadrupole ESI-MSCollision gas

into Q2

Q1 Q3electrosprayion beam

detector

m/z

%

MSM+

M+ frag

Q1Q2 and Q3 set to allow all ions to pass

M+

M+ frag

Q1

M+

Q2fragmentation

Ar

(collison gas)

Q3daughter fragment ions

MS3

M+

M+ frag

Q1

M+ frag

Q2fragmentation

Ar

granddaughter fragment ions

Q3

mass spectrum

m/z

% M+ fragM+

M+

m/z

% M+ frag

daughter fragment ions

M+ frag

granddaughter fragment ions

A triple-quadrupole ESI mass spectrometer possesses ion selection and fragmentation capabilities. Each quadrupole has a separate function: The first quadrupole (Q1) is used to scan across a preset m/z range or to select an ion of interest. The second quadrupole (Q2), also known as the collision cell, transmits the ions while introducing a collision gas (argon) into the flight path of the selected ion, and the third quadrupole (Q3) serves to analyze the fragment ions generated in the collision

cell (Q2). An MS3 experiment can be performed if a daughter fragment ion is generated in electrospray ionization, i.e., M+

frag. Q1 can be used to select the daughter ion, Q2 will generate granddaughter fragment ions, and Q3 will mass analyze for the granddaughter fragment ions.

(Based on G. Siuzdak, 1996)

M+

MS2

Positive Mode Fragmentation


450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/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

732.7

760.8

663.6563.7

730.7 786.8718.7

680.6647.6704.7 788.9

585.6561.7 810.8468.4490.4945.0619.7 813.8413.3

838.9 966.9943.0 1076.0 1174.2994.0 1133.0

ESI+

Up to 135 PLs

700 710 720 730 740 750 760 770 780 790 800m/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

732.7

760.8

758.8733.7

761.8

734.7730.7 786.8

718.7782.7746.7

754.7 787.8768.8762.7704.7 780.7 788.9719.8706.7 735.7 747.8744.8

770.7 794.8736.8 772.7 796.8748.7721.7 789.8707.7 766.7 774.8716.7 728.7708.7

ESI+

Up to 86 PLs

PCphosphatidylcholine

PCePlasmanyl phosphocholine

PCpPlasmenyl phosphocholine

O P

O

O

CH2

CH

CH2

RO

O

OCH2CH2N CH3

CH3

CH3

O

O P

O

O

CH2

CH

CH2

RO

O

OCH2CH2N CH3

CH3

CH3

Phosphatidylcholines

O P

O

O

CH2

CH

CH2

RO

O

OCH2CH2N CH3

CH3

CH3

O

HC

CH

CH2

HN

P

O

O

OCH2CH2N CH3

CH3

CH3

OH

O

Sphingomyelin

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce

184.1

730.5

695.2689.7494.7 614.3261.0 656.6529.7450.3124.8 219.0 547.4147.0 391.1306.4 859.8735.6 819.4338.6 782.7 895.5

M+1

HO P

O

OH

OCH2CH2N CH3

CH3

CH3

Loss of PC headgroup

PEphosphatidylethanolamine

PEePlasmanyl phosphoethanolamine

PEpPlasmenyl phosphoethanolamine

O P

O

HO

CH2

CH

CH2

RO

O

O

OCH2CH2NH2

O

CH2

CH

CH2

RO

O

P

O

HO

OCH2CH2NH2

O

CH2

CH

CH2

RO

O

P

O

HO

OCH2CH2NH2

Phosphatidylethanolamines

100 200 300 400 500 600 700 800 900 1000 1100 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce740.4

599.6

681.3313.4184.0 723.2495.0 555.4295.2 459.6145.2 332.0 665.5268.9

754.579.190.3 898.1 1101.81133.2929.6 1036.4848.4

M+1

Loss of PE headgroup

O P

O

OH

OCH2CH2NH2

H2C

CH

H2C

RO

599.6

OR

Negative Mode Fragmentation


P

O

OH

OCH2CHCH2OH

OH

O

CH2

CH

CH2

RO

OR

O P

O

OH

O(OH)5

CH2

CH

CH2

RO

OR

P

O

OH

OCH2CHCO

NH3

O

CH2

CH

CH2

RO

OR

O

O P

O

HO

CH2

CH

CH2

RO

OR

OH

O P

O

HO

OCH2CH2NH2

CH2

CH

CH2

RO

OR

PE

PG PS

PI

GPA

PE

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce714.5

253.4

281.2

478.3450.2418.2306.3 662.5488.8 757.8539.8 678.7620.8582.0391.1143.3 729.3323.2196.1 223.680.1 771.7 850.0 883.5822.998.0

34:2 PE

16:1

18:1

M-1

6:1

M-1

8:1

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce784.3

697.6

391.2

734.7

417.3 441.4 503.0304.9 752.8281.3 346.1

255.3 479.5528.0

672.4153.3 652.3221.0 957.6575.2 997.0796.0 895.683.335.0 851.4

P

O

OH

OCH2CHCO

NH3

O

CH2

CH

CH2

RO

OR

O88 36:3 PS

20:3

M-8

8-20

:3

M-8

8-16

:0

16:0

M-8

8

18:1

18:2

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce153.0

435.4

522.4

281.1

492.5

487.6195.7 253.0

283.3171.3 237.8

331.8212.5 677.6530.063.9 116.3 373.9 575.8 722.7 748.4 844.7796.5621.8 863.6411.9

M-8

8-18

:1

18:1

M-8

8

M-1

8:1

18:1 LPS

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce909.4

283.4581.0

241.1 419.2 599.0327.1

603.3 747.3463.3331.4 641.3417.5 530.6 811.443.3 700.6 905.6178.3 915.9 970.2152.6 786.6115.2

O P

O

OH

O(OH)5

CH2

CH

CH2

RO

OR

259

259-

H2O

18:0

22:6

M-2

2:6-

163

M-2

2;6

40:6 PI

163

Glycerophospholipid Fragmentation Library

Construct a working phospholipids fragmentation library for B-lymphocytes (subsequently refocused by Steering Cmte to

WEHI-231 cells).

neg pos neg pos neg pos400 500 600401 501 media 601 media402 502 media 602 media403 503 603 media404 504 604 media405 505 605 media406 506 media 606407 507 607408 media 508 608409 16:0 LGPA 509 18:1 LPG 609410 510 610 media411 511 18:0 LPG 611412 media 512 media 612 media413 513 613 media414 514 614415 media 515 615 media416 516 616417 media 517 617 media media418 518 618419 519 619 20:4 LPI420 520 media 620421 521 621 20:3 LPI422 522 18:1 LPS media, 18:1 LPC 622423 523 623424 media 524 media, 18:0 LPS 18:0 LPC 624425 525 625 media426 526 626 media427 527 627 media428 528 628 media429 529 629 media430 530 630 media431 531 media 631 media432 532 632 media433 533 633434 534 634 media435 535 635436 536 636437 media 537 637438 media 538 638 media439 media 539 639440 media 540 640441 media 541 641442 542 642443 543 643444 544 20:4 LPS 644445 545 645446 546 20:3 LPS 646447 547 647448 548 648449 media 549 649450 16:1 LPE 550 20:1 LPS solvent 650

neg pos neg pos neg pos451 551 651 media452 16:0 LPE 16:1LPE 552 652 media453 553 653 media454 media, 16:0 LPE 554 654 media455 555 655 media456 556 656 media457 557 657 media458 558 658 media459 559 659 media460 560 660 media461 561 661462 562 662 30:0 PE media463 563 663 media464 564 media 664 28:0 PCe465 media 565 665466 16:0 PE std 566 666467 16:0 PE std 567 667468 16:0 PE std 568 22:6 LPS 668469 16:0 PE std 569 16:1 LPI 669470 570 22:5 LPS media 670471 571 16:0 LPI 671 34:2 PA472 572 22:4 LPS 672473 573 673 34:1 PA474 574 674 32:1 PEe a/o 32:0 PEp 32:1PEp a/o 32:2 PEe475 575 675 media476 576 676 media, 32:0 PEe 28:1 PC, 32:1 PEe477 577 677 media478 18:1 LPE 578 678 media 32:0 PEe,28:0 PC479 579 679 media480 18:0 LPE 18:1 LPE 580 680 media media481 581 681 media media482 18:0 LPE 582 682 media483 16:0 LPG 583 683484 584 media 684485 585 685486 586 media 686487 587 687488 588 688 32:1 PE489 589 media 689490 media 590 690 32:0 PE 32:1 PE, 30:0PCp a/o 30:1 PCe491 media 591 691 media492 592 692 32:0 PE, 30:0 PCe493 593 693494 16:1 LPS 16:1 LPC 594 694495 media 595 695496 16:0 LPS 16:0 LPC 596 696497 media 597 18:1LPI 697498 598 698499 599 media, 18:0 LPI 699 36:2 PA

neg pos neg pos neg pos neg pos700 30:3 PC 800 38:1PCp a/o 38:2 PCe 900 1000701 media, 36:1 PA media, 16:1 SM 801 901 1001702 media 30:2 PC 802 902 1002703 media 16:0 SM 803 903 1003704 media 30:1PC 804 38:0 PCe 904 1004705 media 805 32:2PI 905 1005706 media 30:0PC 806 38:6PS 38:6 PC 906 1006707 media 807 32:1PI 907 1007708 808 38:5 PS solvent,38:5PC 908 1008709 809 32:0 PI 909 40:6PI 1009710 810 38:4PS media,38:4 PC 910 1010711 811 911 40:5 PI 1011712 34:3 PE 34:4 PE 812 38:3 PS 38:4PS,38:3PC 912 1012713 813 913 40:4 PI 1013714 34:2 PE 34:3 PE 814 38:2 PS 38:2 PC 914 1014715 815 915 1015716 34:1PE 34:2 PE, 32:1PCp a/o 32:2 PCe 816 38:1 PS 38:1 PC 916 1016717 817 917 1017718 34:0PE 34:1PE 818 media 40:6PCp a/o 38:0 PC 918 1018719 32:1PG 819 media 919 1019720 32:0 PCe,34:0PE 820 media 40:5PCp a/o 40:6 PCe 920 1020721 32:0 PG 821 media 921 1021722 36:4PEp 822 media 40:4PCp a/o 40:5 PCe 922 1022723 38:4GPA media 823 923 1023724 824 40:3PCp a/o 40:4 PCe 924 1024725 825 925 1025726 media media 826 40:2PCp a/o 40:3 PCe 926 1026727 media media 827 927 1027728 media, 36:1 PEp, 36:2PEe 32:3 PC, 36:2PEp a/o 36:3 PEe 828 40:1PCp a/o 40:2 PCe 928 1028729 829 929 1029730 32:2 PS, 36:0PEp, 36:1 PEe 32:2 PC, 36:1PEp a/o 36:2 PEe 830 40:0PCp a/o 40:1 PCe 930 1030731 831 931 1031732 32:1PS 32:1PC 832 40:0 PCe 932 1032733 833 34:2PI 933 media 1033734 32:0 PS 32:0PC 834 40:6PS 40:6 PC 934 media 1034735 media 835 34:1PI 935 media 1035736 36:5 PE 836 40:5 PS 40:5 PC 936 1036737 837 937 1037738 36:4PE 34:4PCp,36:5PE 838 40:4 PS 40:4 PC 938 1038739 839 939 1039740 36:3 PE 36:4PE 840 940 1040741 841 941 1041742 36:2PE 36:3 PE, 34:2PCp a/o 34:3 PCe 842 media 40:2 PC 942 1042743 843 943 1043744 36:1 PE 36:2 PE, 34:1PCp a/o 34:2 PCe 844 media 944 1044745 34:2PG 845 945 1045746 38:6 PEp 36:1PE, 34:0PCp a/o 34:1 PCe 846 media 946 1046747 34:1PG 847 947 1047748 38:5PEp a/o 38:6 PEe 34:0 PCe 848 948 1048749 849 949 1049750 38:4PEp a/o 38:5 PEe 38:5PEp a/o 38:6 PEe 850 950 1050

neg pos neg pos neg pos neg pos751 851 951 1051752 34:5 PC, 38:4PEp a/o 38:5 PEe 852 952 1052753 853 953 1053754 34:4PC 854 954 1054755 855 955 media 1055756 34:3PC 856 956 media 1056757 857 36:4PI 957 media 1057758 34:2PS 34:2PC 858 958 1058759 859 36:3 PI 959 1059760 34:1PS 34:1PC,34:2PS,media 860 960 1060761 861 36:2PI 961 1061762 34:0PS, 38:6 PE 34:0 PC, 38:0 PEe 862 962 1062763 863 36:1PI 963 1063764 38:5PE 38:6 PE 864 964 1064765 865 36:0PI 965 1065766 38:4PE 38:5PE, 36:4PCp 866 966 1066767 867 967 1067768 38:3 PE 38:4Pe, 36:3PCp a/o 36:4 PCe 868 968 1068769 869 969 1069770 38:3PE 36:2PCp a/o 36:3 PCe 870 970 1070771 871 media 971 1071772 38:1 PE 36:1PCp a/o 36:2 PCe 872 972 1072773 36:2PG 873 973 1073774 40:6PEp 36:0PCp a/o 36:1 PCe 874 974 1074775 36:1 PG 875 975 1075776 40:5PEp a/o 40:6 PEe 36:0 PCe,38:0PE a/o 40:6 Pep 876 976 1076777 877 977 1077778 36:6 PC,40:5PEp a/o 40:6 PEe 878 978 1078779 879 979 1079780 36:5PC 880 980 media 1080781 881 981 1081782 36:4PS 36:4PC,media 882 982 1082783 883 38:5PI 983 1083784 36:3PS 36:3 PC 884 984 1084785 885 38:4PI 985 1085786 36:2PS solvent, 36:2PC 886 986 media 1086787 887 38:3PI media 987 1087788 36:1PS 36:1PC,media 888 988 1088789 889 38:2 PI 989 1089790 40:6 PE, 36:0 PS 40:0 PEe,36:0PC a/o 38:6 PCp 890 990 1090791 891 991 1091792 40:5 PE 40:6 PE, 38:5PCp a/o 38:6 PCe 892 992 1092793 893 993 1093794 media 40:5PE,38:4PCp a/o 38:5 PCe 894 994 1094795 895 995 1095796 40:4PE,38:3PCp a/o 38:4 PCe 896 996 1096797 38:4PG 897 997 1097798 38:2PCp a/o 38:3 PCe 898 998 1098799 899 999 1099

Cellular Lipid Analysis Program

Generate new computer program software to achieve computational analysis of mass spectrometry data to detect

qualitative changes in membrane lipid composition.

Peak Identification Process:• Potential Peaks are identified from raw data as possessing a L-H-L pattern.• This list is then parsed to find peaks with an associated “brother peak” 1

704.0 7621 0

704.1 7933 0

704.2 8664 0

704.3 14565 0

704.4 26336 0

704.5 37461 0

704.6 41652 1

704.7 35631 0

704.8 21724 0

704.9 9646 0

705.0 4242 0

705.1 2801 0

705.2 3827 0

705.3 8082 0

705.4 16250 0

705.5 25010 0

705.6 28309 1

705.7 23551 0

705.8 15342 0

705.9 9338 0

706.0 6556 0

706.1 6139 0

706.2 10805 0

Inte

nsit

y

m/z

m/z Intensity

Mass Spectrometer OutputPeak?

Analysis of Mass Spectrometry Data

Dalton to the right with a smaller intensity.

After peaks are identified we compute the Mean and Standard Deviation of the peak Intensity (or a transformation thereof.)


0.493

-0.023

0.370

Mean

After the peak list has been compiled the mean and standard deviation for this list is computed.For the current data set these values are: Mean = 44168 SD = 109,724.

The peaks are then standardized by calculating the number of deviations they occur above or below the mean. This is a unit-less number, and a peak that has an intensity equal to the mean intensity will receive a score of zero. Note that peaks which occur below the mean will be assigned a negative number. (See Figure 2 for details.)

This process is repeated across all time points and repetitions for the basal and agonist conditions.

Figure 2: Selected transformed peak values in the m/z range of 700 to 725.

Figure 1: Raw intensity data from the Mass spectrometer .

Basal T=1.5 T=1.5 T=1.5 T=1.5 T = 3 T = 3 T = 3 T = 3 T = 6 T = 6

706.6 0.827 0.77 -1.14 0.898 1.1 0.622 1.249 0.066 0.711 -0.23

Basal T = 6 T = 6 T= 15 T= 15 T= 15 T= 15 T=240 T=240 T=240 T=240

706.6 0.352 -0.741 1.031 3.302 1.5 0.346 0.8413 0.377 1.32 -0.786


Group Mean

T = 1.5 T = 6 T = 15 T = 240T = 3

• Plots the points with respect to time and the mean within the observational units.

• Generates Upper and Lower control limits for the means of the observational units. These limits represent the expected variation within the observed means. (Not individual observations!)

• Performs a set of statistical tests looking for “out-of-control” conditions (i.e. non-random variation around the grand mean.)

The signals are then analyzed on a line by line basis to form a profile for a given m/z value.Below is the data from the m/z value of 706.6 in the basal condition. There are four repetitions in each of 5 time points, 1.5, 3, 6, 15, and 240 minutes.

Shewhart Control Chart for m/z = 706.6 Basal Next a Shewhart Control Chart is constructed for the mean of the transformed signal. This chart:

Review of Shewhart Control Theory

Test 1: One point beyond Zone A.Test 2: Two out of three points in

a row in Zone A or beyond.

Test 3: Four out of five points in a row in Zone B or beyond.

Facts about Shewhart Control Charts:

Various statistical tests exist for looking for special cause variation. These include but are not limited to:

Where is the estimate for the population standard deviation and n is the number of repetitions in a group.

The control limits for the mean are calculated with the formula:

The various zones, A, B, and C, are then calculated by dividing the are between the grand mean and the control limits into thirds. The formulas for these zones and the expected percentage of means falling in them are given in the figure to the right.

nX

3

A23187 T=1.5 T=1.5 T=1.5 T=1.5 T = 3 T = 3 T = 3 T = 3 T = 6 T = 6

706.6 3.68 2.49 1.28 0.39 1.25 0.36 3.14 1.61 -0.36 0.94

A23187 T = 6 T = 6 T= 15 T= 15 T= 15 T= 15 T=240 T=240 T=240 T=240

706.6 -0.21 1.63 1.59 -0.26 1.50 1.43 1.33 0.28 -0.78 -0.87


T=1.5 T=6 T=240T=15T=3

Out of Control points. (Rule 2 of 3 in Zone A or beyond)

Data from the m/z value 706.6 for 2H3 cells treated with Ionophore.

The analysis then uses the limits calculated from the basal condition to analyze the stimulated cells. (Assuming the basal data is in control i.e. only random variation present.)

Basal Data Ligand Data

The program looks for patterns in the stimulated case using the limits from the basal condition. Out of control points are marked and cataloged as in the figure at the right.

The results of the analysis are then grouped into arrays giving a comprehensive view of time based lipid changes in the cell.

PIP Standards

Determine feasibility of measuring poly-phosphatidylinositol species as part of the LIPID ARRAYS.

PI

PI-5-P

PI-3-P

PI-4-P

PI-3,4-P

PI-4,5-P

PI-3,4,5-P

PI-3,5-P

PI-3,5-P

PI4K

PI 4-phosphatase

PI3K

PI 3

-pho

spha

tase

PI5KPI 5-phosphatase

PI4K

PI 4-phosphatase

PI4K

PI 4-phosphatase

PI5K

PI3K

PI3K

PI 3

-pho

spha

tase

PI5KPI 5-phosphatase PI3K

PI 3

-pho

spha

tase

PI5KPI 5-phosphatase

(PTE

N)

Poly-phosphatidylinositols

880 900 920 940 960 980 1000 1020 1040

m/z

23

0

5

10

15

100

0

20

40

60

80

41

0

10

20

30

Rel

ativ

e A

bund

ance

72

0

20

40

60

13

0

5

10

1045.3

965.3

1049.7889.3 1043.3

967.51051.2

963.5 1019.2 1032.3995.4 1005.3969.3890.3 950.7915.2 942.3 988.3928.1907.1879.3 971.4961.6

1049.2

1050.1

889.41051.2969.4

890.2 1045.3991.2 1052.4965.4 971.5 1023.3952.7911.3 1001.1 1006.7884.1 893.2 923.6 939.3

1045.4

1046.3

1044.3 1047.3965.3 1032.31017.4894.5967.6943.4 1005.1963.2 993.4937.7879.0 923.3904.4 912.2 977.5

965.3

966.3

967.3

937.2 943.3 963.4915.3993.4969.3 982.3878.3 889.3 904.5 997.4927.9 1023.41009.1 1054.21043.3

889.3

890.4885.3 911.2

912.3891.2919.4 1039.4989.2900.7 965.8 1050.0949.5 998.4981.1929.3 1007.6 1014.7

Poly-PIP mixture

32:0 PI-3-phosphate

38:4 PI-4-phosphate

38:4 PI-4,5-phosphate

32:0 PI-3,4,5-phosphate

LIPID ARRAYS

Initiate LIPID ARRAYS on ligand stimulated WEHI-231 cells.

cAMP

2MA, PGE2, TER, CGS

P-protein

CD40L, IL4, IL10, INF

Calcium

AIG, MIP3a, SLC

LPA

BLC, ELC, SDF1, S1P

Relationship of 15 ligands with clear responses in B cells

Sternweis

Regulation of B cell activation by antigen-antibody complexes and Ig Fc receptors

Antigen-antibody complexes can simultaneously bind to membrane Ig (via antigen) and the FcRIIB receptor via the Fc portion of the antibody. As a consequence of this simultaneous ligation of receptors, phosphatases associated with the

cytoplasmic tail of the FcRIIB inhibit signaling by the B cell receptor complex and block B cell activation. Fc, crystallizable fragment; FcRIIB, Fcreceptor II; Ig, immunoglobulin.

Lipid Arrays from WEHI-231 Cells treated with Anti-IgM (ELC030121A – 0219A)

Negative Mode Positive Mode

WEHI Cell Lipid Arrays Summary(Negative Mode)

WEHI Cell Lipid Arrays Summary(Positive Mode)

40:2 PCp 40:3 PCe

38:1 PCp 38:2 PCe

36:0 PCp 36:1 PCe

32:0 PE 30:0 PCe

40:2 PCp 40:3 PCe

28:1 PC 32:1 PEe 36:2 PC 40:6 PC

32:1 PE 30:0 PCp 30:1 PCe

36:1 PE 34:0 PCp 34:1 PCe

40:2 PCp 40:3 PCe

36:1 PCp 36:2 PCe

34:2 PE 32:1 PCp 32:2 PCe

40:3 PCp 40:4 PCe

36:0 PCp 36:1 PCe

36:2 PE 34:1 PCp 34:2 PCe

36:6 PC 40:5 PEp 40:6 PEe

40:3 PCp 40:4 PCe

28:1 PC 32:1 PEe

34:0 PC 38:0 PEe

40:5 PCp 40:6 PCe

36:1 PCp 36:2 PCe

36:2 PE 34:1 PCp 34:2 PCe

36:1 PE 34:0 PCp 34:1 PCe

36:1 PE 34:0 PCp 34:1 PCe

32:1 PE 30:0 PCp 30:1 PCe

38:1 PCp 38:2 PCe

32:1 PE 30:0 PCp 30:1 PCe

40:4 PE 38:3 PCp 38:4 PCe

32:2 PC 36:1 PEp 36:2 PEe

40:4 PE 38:3 PCp 38:4 PCe

40:0 PEe 36:0 PC

38:6 PCp38:3 PE

36:2 PCp 36:3 PCe

36:0 PCe 38:0 PE

40:6 PEp

36:2 PE 34:1 PCp 34:2 PCe

36:2 PC 30:1 PC 34:1 PE 40:4 PC

36:4 PC 34:3 PC 36:5 PC 40:6 PC

38:4 PC 34:2 PC 36:1 PC 30:3 PC

34:0 PCe 36:2 PC 38:6 PC

38:6 PE 36:4 PC 38:0 PCe

40:3 PCp 40:4 PCe

40:5 PCp 40:6 PCe

34:1 PC 34:2 PS

40:6 PCp 38:0 PC

40:1 PCp 40:2 PCe

34:1 PC 34:2 PS 38:0 PCe

38:3 PE 36:2 PCp 36:3 PCe

32:0 PCe 34:0 PE

32:2 PC 36:1 PEp 36:2 PEe

38:2 PCp 38:3 PCe

40:0 PCp 40:1 PCe

38:3 PE 36:2 PCp 36:3 PCe

40:4 PE 38:3 PCp 38:4 PCe

40:5 PE 38:4 PCp 38:5 PCe

38:1 PC32:0 PEe 28:0 PC

38:4 PE 36:3 PCp 36:4 PCe

36:1 PE 34:0 PCp 34:1 PCe

30:0 PC32:0 PCe 34:0 PE

40:5 PE 38:4 PCp 38:5 PCe 30:1 PC

36:1 PCp 36:2 PCe

38:6 PE 36:3 PC 32:1 PC 38:6 PE

34:2 PC 36:5 PC

30:1 PC

36:1 PC

36:3 PC

38:5 PC

38:6 PC

Time = 240minTime = 1.5min Time = 3min Time = 6min

Sig

nif

iga

nt

Time = 15min

De

cre

asi

ng

De

cre

asi

ng

Hig

hly

Sig

nif

iga

nt

POS

CD40 (40L)Anti-IgM (AIG) BLC

WEHI Cell Lipid Arrays(Negative Mode)

LC Class Separation

Future Aim: Transition from qualitative to quantitative analysis.

Brugger et al.PNAS, 1997

Phosphatidylcholine Class Separation

Varian 240 LCPolymer Labs 1000 ELSDPhenomenex Luna Si column0.3 mL/min70%ACN, 20%IPA, 10%H2O, 25mM ammonium formate

500 600 700 800 900 1000 1100 1200

m/z

100

0

10

20

30

40

50

60

70

80

90

37

0

5

10

15

20

25

30

Rel

ativ

e A

bund

ance

732.5

760.6

782.5718.5

704.4408.2 550.7 808.5 822.5678.4 880.7522.5 634.5424.3 1137.0934.6 996.6 1189.01110.91024.9

563.5

732.4

760.4

561.5

647.3535.4 663.4 786.5585.4 944.6718.4

801.4509.5

812.5 942.6496.3 961.6866.7 1000.7408.1 1041.8 1087.7 1134.7

PC fraction

Total Extract

600 650 700 750 800 850 900 950 1000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce790.3

818.4

816.4776.3

819.4

844.4

764.3

846.4762.4

830.5750.4742.4 872.3

886.6627.4 914.5716.3694.2651.3 1000.6940.4613.3 956.5 972.3681.4

PC Fraction +NH4OAc31-38 min

ESI (-)

compound std1 std2 std3 std4phosphatidylcholine 25:0 31:1 37:4 43:6phosphatidylethanolamine 25:0 31:1 37:4 43:6phosphatidylglycerol 25:0 31:1 37:4 43:6phosphatidylinositol 25:0 31:1 37:4 43:6PI-monophosphate(PIP) 37:4PI-diphosphate(PIP2) 37:4PI-triphosphate (PIP3) 37:4phosphatidylserine 25:0 31:1 37:4 43:6lysophosphatidic acid 13:0 17:1 19:3 23:4phosphatidic acid 25:0 31:1 37:4 43:6cardiolipin 57:0 61:1 65:3 69:4lysophosphatidylcholine 13:0 17:1 19:3 23:4diacylglycerol 25:0 31:1 37:4 43:6

Isotopic Standards for Quantitative Phospholipid Analysis

Ligand

cAMP

The Cell: A lipocentric view

GPCR

Calcium

Cell morphology (microscopy)

Phagocytosis

Pinocytosis

Chemotaxis

Secretion

Phosphoproteins

Gene arrays

LipidSignaling

32:1 PEp32:1 PE34:0 PE34:2 PE36:5 PE

40:6 PS40:7 PS

36:4 PI38:4 PI

16:0 LPE, 16:1 LPE16:0 LPE, 16:1 LPE16:0 LPE16:1 LPE16:1 LPE

22:6 LPS22:6 LPS

20:4 LPI20:4 LPI

34:1 PC 34:1 GPA

30:0 PC 16:0 LGPA

32:0 PCp 16:0 LGPA

32:1 PC 16:0 LGPA

34:0 PCe 16:0 LGPA

Modeling of lipid signaling: precursor-product relationships

Rationale for dual-ligand screens

• Ligand A many lipid changes• Ligand B little or no lipid changes

Conclusion: Outcomes of dual-ligands are complex

PC

PA DAG

PI, PG

PE, PS ,PC

LPP

GPCRB

X

PO42-

Ligand B

GPCRA

Ligand A

Modeling lipid pathways: AIG Arrays

Apoptosis

Substrate-Product

36:4 PC 20:4 lysoPC

16:0 ffa

PS lysoPS

RNAi

PLA1/PLA2

LIPID MAPS Glue Grant, Ed Dennis P.I.

Macrophage lipids

Bioinformatics Neutral Lipids

Glycolipids

Sterols

Fatty Acids/Eicosanoids

Phospholipids

Sphingolipids/Gangliosides

Synthetic Lipid Design

•Mike VanNieuwenhze - UCSD•Dale Boger – Scripps Inst.•Camille Falck- UTSW•Walter Shaw - Avanti Polar Lipids

•Shankar Subramaniam - UCSD •Robert C. Murphy – University of Colorado at Denver

•Chris Raetz – Duke Med. Cntr.

•David W. Russell - UTSW •Alfred H. Merrill - Georgia Tech.

•H. Alex Brown – Vanderbilt Med. Cntr.

•Edward Dennis – UCSD

Macrophage Core Laboratory

•Chris Glass - UCSD

Expanded analysis of Cellular Lipids

Lipid Class Species

Robert Murphy/UC-Denver Neutral Lipids MAG, DAG, TAG.

Ed Dennis/ UCSD Fatty acids/Eicosanoids Prostanoids; hydroxyl- and hydroperoxy- eicosaenoicacids, and leukotrienes; and epoxyeicosatrienoic acids.Free fatty acids, fatty acid amides.

Alex Brown/ Vanderbilt Phospholipids PC, PE, PG, PS, PI (and polyphospho derivatives), PA,cardiolipin, lysophospholipids, plasmalogens and otherether-linked phospholipids, prostanoid containing phospholipids.

Al Merrill/ GA Tech Sphingolipids/Gangliosides Sphingomyelin, glycosphingolipids, ceramides, sphingosine.

David Russell/ UTSW Sterols Isoprenoids, sterols, and bile acids.

Chris Raetz/ Duke Glycolipids Polyisoprene-linked phosphate sugars, certain fat soluble vitamins and quinines , and the glycolipid precursors of the PI glycans.

PI/ Institution

RAW264.7 macrophage spectra (positive mode)

Resting LPS

030418 RMJ001 Sample 5 LPS Pos AV:52 NL: 9.48E6

450 500 550 600 650 700 750 800 850 900m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

760.7

663.6563.6

732.7786.7

758.7703.6

664.6787.7

680.6585.6

704.7

813.8561.5

535.5 619.7 815.8647.5520.5496.4 836.8

641.7591.6494.4840.8 864.8

030418 RMJ001 Sample 2 no LPS Pos AV:53 NL: 1.24E7

450 500 550 600 650 700 750 800 850 900m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

760.7

786.7732.7663.5

758.7

703.7563.6

788.7680.6

808.7585.6 706.6

814.7520.4 561.5

834.7647.6509.5 619.7496.4840.7 862.8 890.8

Primary murine macrophage spectra (negative mode)

Vehicle control Acetylated LDL LoadedFazio005sample1 vehicle control Neg AV:52 NL: 3.01E6

500 600 700 800 900 1000 1100 1200m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

819.4773.5

613.3

885.4

711.2671.2

585.3748.4

865.4473.0

529.3

509.2 1049.4915.5557.3

919.4 993.41175.71085.3

Fazio004 Sample 8 loaded acLDL Neg AV:53 NL: 1.31E6

500 600 700 800 900 1000 1100 1200m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

489.0

613.3

641.4

885.4

771.3

786.3659.3585.3 807.3715.4

697.2503.0 865.3

743.2

539.3

1073.7907.7 1045.6953.71085.7 1136.7

Lipofectamine

Lipid-mediated transfection: time course of membrane recovery?

Construct a working phospholipids fragmentation library for RAW 264.7* cells (* or alternative cells selected by AfCS Steering Committee).

Begin adaptation of phospholipid headgroup classes (i. e. PC, DAG) to quantitative analysis by stable isotope dilution (LIPID MAPS).

Achieve quantitative poly-phosphatidylinositol species analysis for selective LIPID ARRAYS.

Adapt cellular lipid analysis program software to quantitative computational analysis of mass spectrometry data. Work closely with Bioinformatics and Data Analysis Group to convert phospholipid species into numerical format compatible with Oracle format to facilitate cluster analysis and modelling.

Initiate LIPID ARRAYS in RAW264.7 or primary macrophages cells using ligand list generated by Steering Cmte.

Based on choice of macrophage processes (i.e. chemotaxis, secretion) use cluster analysis to identify phospholipid signaling species of interest.

Lipidomics Lab

Specific Aims 2003-04

Multiplexed lipid arrays of ligand-induced changes The Alliance for Cell Signaling 2003 Meeting-...

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