2
Multiplexed Glycerophospholipid Analysis of AfCS Ligand Screen Stephen B. Milne, Jeffrey S. Forrester, Pavlina T. Ivanova, Michelle D. Armstrong and H. Alex Brown AfCS Lipidomics Lab, Department of Pharmacology & VICB, Vanderbilt University Medical Center Introduction. The primary goal of the Alliance for Cellular Signaling (AfCS) is to determine how cells process signaling information in a context dependent manner. As part of its effort to elucidate the signaling network in the RAW 264.7 macrophage cell line, the AfCS is conducting a broad based ligand screen to identify which ligands, either alone or in combination, are most relevant to the regulation of the behavior of these cells. A collection of information-rich assays has been selected to sample both the signaling capability of the cell and the complexity of the response, as well as to identify unique and interactive inputs. This phase of experimentation is not intended to yield mechanistic information, but rather to determine the connectivity of the network as measured through various mathematical criteria, including Boolean network analysis and clustering algorithms. The Lipidomics laboratory has been participating in this ligand screen by creating multiplexed lipid arrays that highlight changes in cellular phospholipids in RAW 264.7 cells after stimulation. We have analyzed six individual and six dual ligand combinations, and the resulting lipid arrays are presented here. Our results suggest both common and differential activation of signaling pathways involving bioactive lipid effectors by the various ligands studied. In addition, we present an example of a synergistic ligand interaction. Lipid arrays were created using methods described in the AfCS research report, Milne et al., (2003) AfCS Res. Reports (www.signaling-gateway.org/reports). Each lipid array represents a summary created from a minimum of five individual arrays. Data for the individual arrays were collected at five time points (0.5, 2, 7, 15, and 30 min.) each repeated in triplicate. Statistically significant changes in lipid species between basal and stimulated states are indicated with colors: red (relative increase), blue (relative decrease), and green (no change). Figure 2. Sphingosine-1- phosphate (S1P) Stimulation. Lipid array for RAW 264.7 cells stimulated with 1 M S1P. Note the increase [shown in panels (2) & (3)] of several as yet unidentified lipid species. (C*) Data showing measurement of intracellular free Ca 2+ concentrations after addition of 30 M S1P. Figure 3. Platelet Activating Factor (PAF) Stimulation. Lipid array for RAW 264.7 cells stimulated with 100 nM PAF. Note the slight increase in phosphatidic acid (PA) species (1) - (3) and the decrease in phosphatidylinositol (4) & (5). (C*) Data showing measurement of intracellular free Ca 2+ concentrations after addition of 100 nM PAF. Figure 4. PAM2CSK4 (P2C) Stimulation. Lipid array for RAW 264.7 cells stimulated with 1 M P2C. Note the significant decreases in phosphatidyl-inositol (PI) lipid species containing highly unsaturated fatty acids and the increase in mono- unsaturated and saturated species. (C*) Data Figure 1. Interleukin-4 (IL4) Stimulation. Lipid array for RAW 264.7 cells stimulated with 500 pM IL4 (AfCS 3- Letter code: I04). Note the increase [shown in panels (1) & (2)] of several species of ceramides. (C*) Data showing measurement of intracellular free Ca 2+ concentrations in RAW 264.7 cells after addition of 250 pM IL4. S1P w ith H ighlights (N egative M ode Ionization,Five D ata sets) 902.51 0 0 1 0 0 0 -1 -1 -1 0 904.60 0 0 0 1 3 0 0 0 0 2 0 0 0 0 1 907.50 0 0 0 1 3 0 0 -1 1 4 40:6PI 909.41 -1 -1 -1 -1 -1 Tim e (m in.) Lipid m/z 0.5 2 7 15 30 929.48 0 0 0 0 0 0 0 0 3 0 0 1 2 932.00 0 0 0 0 0 0 0 1 3 0 0 0 2 934.16 -1 -1 -1 -1 -1 0 0 0 2 0 0 0 3 936.57 0 0 0 1 3 0 0 1 2 938.56 0 0 0 0 0 Tim e (m in .) 882.40 0 0 0 1 1 38:5PI 883.36 0 0 0 0 -1 884.36 0 0 0 0 -1 38:4PI 885.38 -1 -1 -1 0 -1 886.38 0 0 0 0 -1 38:3PI 887.39 -1 -1 -1 0 -1 0 0 0 0 -1 0 -1 -1 -1 -1 890.43 0 0 0 0 -1 -100 0 100 200 300 400 500 600 Time (s) 700 [Ca 2+ ](nM ) 30 M S phingosine-1-phosphate 0 20 40 60 80 100 120 1 2 3 1 2 3 C* * Data obtained from the AfCS Data C enter PA F w ith H ighlights (N egative M ode Ionization,Six D ata sets) 0 -1 1 1 0 38:5P I 883.50 -1 -1 -2 0 -2 -1 -1 -1 -1 -1 0 -1 -3 -1 -1 886.53 -1 0 -2 -1 -2 0 -1 -2 -1 -1 -1 1 -2 -2 -2 38:2P I 889.55 0 0 -2 -1 -2 0 0 -2 0 0 891.58 -1 0 -1 -1 -1 Lipid m/z 0.5 2 7 15 30 1 1 1 4 1 0 1 0 2 0 0 1 0 1 0 36:4P I 857.50 -1 0 -1 0 -2 0 0 0 0 0 0 0 -2 0 -1 0 0 0 0 0 0 0 -1 -1 -1 862.52 0 0 -2 0 -1 36:1P I 863.53 0 0 -1 0 -1 0 0 0 0 -1 865.54 -1 0 0 0 -1 Tim e (m in.) Lip id m/z 0.5 2 7 15 30 34:2P A 671.41 0 2 3 1 1 -1 -1 1 0 0 0 0 1 0 0 0 0 -1 0 0 2 3 3 3 2 696.47 0 1 1 -1 0 36:3P A 697.43 0 1 2 1 1 -1 -1 1 -1 0 0 0 1 1 0 700.43 0 0 1 -1 1 [C a 2+ ](nM) 100 nM P lateletActivating Factor -100 0 100 200 300 400 500 600 Tim e (s) 700 0 50 100 150 200 250 1 2 3 4 5 1 2 3 5 4 C* * Data obtained from the AfC S Data C enter P2C w ith H ighlights (N egative M ode Ionization,Six D ata sets) 881.53 0 1 0 0 0 0 -1 -1 0 -2 -2 0 -1 -1 -4 884.52 -2 0 -1 -1 -5 -2 -1 0 -1 -5 -2 0 -2 -1 -4 -3 1 -1 0 -1 -2 1 -2 -1 -2 38:2PI 889.55 -1 0 -1 -1 -1 -3 2 -2 0 1 -2 1 -1 0 0 892.60 -1 0 0 0 1 893.60 0 0 0 0 0 Tim e (m in.) L ipid m/z 0.5 2 7 15 30 -1 0 0 -1 -3 856.54 0 0 0 0 0 -1 -1 0 0 -6 858.52 0 0 0 0 -5 -1 0 0 0 -1 860.53 1 -1 0 0 0 -2 0 0 0 0 862.53 -1 0 0 0 0 -1 0 1 1 4 -1 0 1 0 3 865.54 0 0 1 1 4 866.56 0 -1 0 0 0 0 0 0 0 910.56 -1 0 0 -1 -1 0 0 1 0 0 0 0 0 2 0 1 2 0 0 1 0 0 0 0 1 0 916.59 0 0 0 1 1 0 -1 1 0 0 0 0 0 0 -1 -1 -1 0 920.67 -1 1 -1 0 -2 Tim e (m in .) -100 0 100 200 300 400 500 600 Time (s) 350 nM PAM 2CSK4 0 20 40 60 80 100 [Ca 2+ ](nM ) 700 1 2 3 1 2 3 C* * Data obtained from the AfCS Data C enter 0 1 1 1 1 1 0 3 1 3 0 0 0 0 1 2 1 2 1 1 0 0 0 0 0 590.33 0 1 3 2 1 34:2D AG 591.32 0 0 1 0 0 C20:0S 592.36 0 0 0 2 1 34:1D AG 593.34 -1 1 1 1 0 Lipid m/z 0.5 7 15 30 615.30 0 0 0 0 0 0 1 2 2 1 30:1PA, 20:5LPI, 0 1 1 1 0 2 0 3 2 2 20:4LPI, 30:0PA, 0 1 1 1 1 0 1 0 2 0 20:3LPI, 36:1D AG 621.35 1 1 1 1 0 Tim e (m in.) 0 0 0 0 0 36:4P E, -1 -1 -2 -1 0 739.32 0 0 0 0 0 0 0 0 0 0 32:1P C (Cl), 38:4PE 766.32 -1 -2 -1 -3 0 767.32 -1 0 -1 -1 0 -100 0 100 200 300 400 500 600 700 [Ca 2+ ](nM ) 250 pM Interleukin-4 0 20 40 60 80 100 1 2 3 4 Tim e (s) 1 2 3 4 IL4 w ith H ighlights (N egative M ode Ionization,Five D ata sets) C * * Data obtained from the AfC S Data C enter

Multiplexed Glycerophospholipid Analysis of AfCS Ligand Screen

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Multiplexed Glycerophospholipid Analysis of AfCS Ligand Screen Stephen B. Milne, Jeffrey S. Forrester, Pavlina T. Ivanova, Michelle D. Armstrong and H. Alex Brown AfCS Lipidomics Lab, Department of Pharmacology & VICB, Vanderbilt University Medical Center. Introduction. - PowerPoint PPT Presentation

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  • Multiplexed Glycerophospholipid Analysis of AfCS Ligand ScreenStephen B. Milne, Jeffrey S. Forrester, Pavlina T. Ivanova, Michelle D. Armstrong and H. Alex BrownAfCS Lipidomics Lab, Department of Pharmacology & VICB, Vanderbilt University Medical Center

    Introduction. The primary goal of the Alliance for Cellular Signaling (AfCS) is to determine how cells process signaling information in a context dependent manner. As part of its effort to elucidate the signaling network in the RAW 264.7 macrophage cell line, the AfCS is conducting a broad based ligand screen to identify which ligands, either alone or in combination, are most relevant to the regulation of the behavior of these cells. A collection of information-rich assays has been selected to sample both the signaling capability of the cell and the complexity of the response, as well as to identify unique and interactive inputs. This phase of experimentation is not intended to yield mechanistic information, but rather to determine the connectivity of the network as measured through various mathematical criteria, including Boolean network analysis and clustering algorithms. The Lipidomics laboratory has been participating in this ligand screen by creating multiplexed lipid arrays that highlight changes in cellular phospholipids in RAW 264.7 cells after stimulation. We have analyzed six individual and six dual ligand combinations, and the resulting lipid arrays are presented here. Our results suggest both common and differential activation of signaling pathways involving bioactive lipid effectors by the various ligands studied. In addition, we present an example of a synergistic ligand interaction. Lipid arrays were created using methods described in the AfCS research report, Milne et al., (2003) AfCS Res. Reports (www.signaling-gateway.org/reports). Each lipid array represents a summary created from a minimum of five individual arrays. Data for the individual arrays were collected at five time points (0.5, 2, 7, 15, and 30 min.) each repeated in triplicate. Statistically significant changes in lipid species between basal and stimulated states are indicated with colors: red (relative increase), blue (relative decrease), and green (no change).Table 1. Results of RAW 264.7 StimulationTable 1. Results of RAW 264.7 StimulationTable 1. Results of RAW 264.7 StimulationTable 1. Results of RAW 264.7 StimulationFigure 2. Sphingosine-1-phosphate (S1P) Stimulation.Lipid array for RAW 264.7 cells stimulated with 1 mM S1P. Note the increase [shown in panels (2) & (3)] of several as yet unidentified lipid species. (C*) Data showing measurement of intracellular free Ca2+ concentrations after addition of 30 mM S1P.Figure 3. Platelet Activating Factor (PAF) Stimulation.Lipid array for RAW 264.7 cells stimulated with 100 nM PAF. Note the slight increase in phosphatidic acid (PA) species (1) - (3) and the decrease in phosphatidylinositol (4) & (5). (C*) Data showing measurement of intracellular free Ca2+ concentrations after addition of 100 nM PAF.Figure 4. PAM2CSK4 (P2C) Stimulation.Lipid array for RAW 264.7 cells stimulated with 1 mM P2C. Note the significant decreases in phosphatidyl-inositol (PI) lipid species containing highly unsaturated fatty acids and the increase in mono- unsaturated and saturated species. (C*) Data showing measurement of intracellular free Ca2+ concentrations after addition of 350 nM P2C.Figure 1. Interleukin-4 (IL4) Stimulation.Lipid array for RAW 264.7 cells stimulated with 500 pM IL4 (AfCS 3-Letter code: I04). Note the increase [shown in panels (1) & (2)] of several species of ceramides. (C*) Data showing measurement of intracellular free Ca2+ concentrations in RAW 264.7 cells after addition of 250 pM IL4.

  • S1P with Highlights

    (Negative Mode Ionization, Five Data sets)

    -100

    0

    100

    200

    300

    400

    500

    600

    Time (s)

    700

    [Ca2+] (nM)

    30 mM Sphingosine-1-phosphate

    0

    20

    40

    60

    80

    100

    120

    1

    2

    3

    1

    2

    3

    C*

    * Data obtained from the AfCS Data Center

    Sheet2

    Lipidm/z0.5271530Lipidm/z0.5271530Lipidm/z0.5271530Lipidm/z0.5271530

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    543.190000038:2PE770.34-1-1-1-2-1985.61112101143.8200000

    20:4LPS, 17:0LPC(Cl)544.23111-1136:3PG771.33-1-100-1986.64-100001144.2611111

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    595.250000040:5PG823.3901-1121028.67000001175.760-1000

    596.2300000824.38110001029.64-1-1-1001176.750-21-10

    34:0DAG, 18:2LPI597.160000040:4PG825.39121201030.66001001177.770-21-10

    598.2300000826.39000001031.01000001178.8100000

    18:1LPI599.2200000827.39120201031.61-101111179.8300000

    600.2911111828.39000101032.11000001180.8401011

    18:0LPI601.2801100829.3911-1101032.64000011181.8410000

    602.3001121830.39000001033.65000001182.8500000

    603.2901111831.39000001034.61000101183.8300000

    22:6LPC(Cl)604.290100140:7PS832.38110-111035.00000001184.8410100

    605.280111134:2PI833.350-10001035.65000001185.3300000

    22:5LPC(Cl)606.350000040:6PS, 37:2PC(Cl)834.37101111036.660-1-2-2-11185.8811011

    607.320000-134:1PI835.360-20001037.65220101186.8500000

    608.290011140:5PS836.36-10-1001038.6800-10-11187.2700000

    609.271101034:0PI837.37000001039.672212-11187.8611111

    22:3LPC(Cl)610.2700-10040:4PS838.38000001040.70000001188.42-1-1-1-1-1

    611.2700000839.370000-11041.69-100-1-11188.8701111

    612.280000-138:6PC(Cl), 40:3PS840.39000001042.72001001189.7900000

    613.2911121841.39000001043.69001-1-11190.1600000

    614.290000038:5PC(Cl)842.39000001044.08000001190.8000000

    615.3001111843.39000001044.71-1-11001191.7800100

    616.301110138:4PC(Cl), 40:1PS844.43121121045.63000001192.7900100

    617.3100000845.4411-1011045.99000001193.8200100

    618.36001-1-238:3PC(Cl)846.45030011046.65000001194.2200000

    619.2900000847.45000011047.61000001194.8500000

    30:1PA, 20:5LPI, 36:3DAG620.3900000848.45010111048.64-1-1-10-11195.8400000

    C22:1S621.350000-1849.45000001049.64-1-1-1-101196.2200000

    20:4LPI, 30:0PA, 36:2DAG622.360000-2850.44110111050.660-20001196.86-2-1-2-2-1

    C22:0S623.3010100851.46000011051.65001001197.8800000

    20:3LPI, 36:1DAG624.2900000852.43000011052.67-1-10-1-11198.87-1-1-100

    Sheet1

    &A

    Page &P

  • PAF with Highlights

    (Negative Mode Ionization, Six Data sets)

    [Ca2+] (nM)

    100 nM Platelet Activating Factor

    -100

    0

    100

    200

    300

    400

    500

    600

    Time (s)

    700

    0

    50

    100

    150

    200

    250

    300

    350

    1

    2

    3

    4

    5

    1

    2

    3

    5

    4

    C*

    * Data obtained from the AfCS Data Center

    Sheet1

    Set: Ligand Screen 2

    Data Sets: ELC040302A, ELC040303A, ELC040309A, ELC040331A, ELC0400406A, ELC0400407A

    Ligand: PAF

    Transformation: Ranks

    Instrument Mode: Negative

    Date of Analysis: 04/26/2004

    Time (min.)

    Lipidm/z0.5271530V2V3V4V5V6V7V8V9V10V11V12V13V14V15V16V17V18V19V20V21V22V23V24V25V26V27V28V29V30V31V32V33V34V35V36V37V38V39V40V41V42V43V44V45V46V47V48V49V50V51V52V53V54V55V56V57V58V59V60V61V62V63V64V65V66V67

    400.20-10000141517839.0717712.233000000151517909.87999.333000000151517559.5337356.733000000141515734.3216066.467000000151515217.3334445.833000000151515635.3674984.10-10000

    401.20000001515189549005.933000000151519194.5338984.6000000151519187.4678963.0670000001515176938024.667000000151516585.66343.133000000151516861.8677204.667000000

    402.22000001515173157281.6000000151517892.3337745.667000000151517724.67379.6000000151516221.66689.267000000151514918.0334648.933000000151515166.0335383.9000000

    403.20110111515193069482.6000000151519548.3339260.4000000151519469.5339336.8000000151518476.69153.533011011151517208.4676984.2000000151517429.4677657.867000000

    404.1800100151517821.87953.733000000151517808.8677714.733000000151517818.3337660.2000000151517070.5337712.4000000151516593.0676464.267000000151516474.66842.267000100

    405.1800000151518148.0678222.40000001515181017989.733000000151518110.0677932.333000000151516620.17278.567000000151516124.45803.7330000001515160876341.933000000

    406.1700000151515887.0675888000000151515663.45650.667000000151515949.0675592.867000000151514613.5335188.117000000151513993.5333881.433000000151514223.1334116.033000000

    16:1LPA407.1600000151518095.2678046.867000000151517639.67632.2100000151517726.8677747.267000000151517439.5337215.733000000151517027.0676945.4000000151515658.0675638.133000000

    408.1700000151515613.5335494.067000000151515203.9335258.933000000151515353.0675236.467000000151515195.44870.667000000151514093.7334022000000151513218.72954.467000000

    16:0LPA409.1700000151519452.4679549.933000000151519521.1339513.2000000151519696.29638.6000000151519399.2679321.467000000151518888.0678966.4000000151518527.6678600.867000000

    410.1800100151517217.4677154.133000000151517353.6677231.933000000151517343.0677352.4000000151517071.4336734.8000000151516359.26547.733100100151516163.7336159.9000000

    411.18-20000151518457.8678481.333000000151518257.68145.8000000151518207.47962.2670-10000151516822.5337143.033000000151516861.0676598.0670-100001515159546150.867000000

    412.2200000151518493.3338431.933000000151518069.0677641.667000000151517361.0677064.733000000151515474.1175792.8000000151515090.9335264000000151515034.8674666.5000000

    413.1800000151519115.89086.6000000151519094.5339073.333000000151518940.68737.067000000151516864.7337581.8000000151516283.4676106.80000001515162396480.267000000

    414.1800000151516588.0676479.4000000151516480.86612.2000000151516365.66228.733000000151514456.5335101.467000000151513633.8673252.4330000001515132933289.967000000

    415.1700000151518914.89014.267000000151519004.88938.133000000151518881.8678762.667000000151517270.1337885.6000000151516698.6676556.7000000151516612.1336858.6000000

    416.19-10000151516503.8676523.2000000151516319.1336468.333000000151516505.46375.1330-10000151514930.75569.067000000151513864.2334371.667000000151513666.7673518.2000000

    417.19-10000151519262.29359.8000000151519264.5339224.067000000151519189.29075.20-10000151518622.49033.6000000151518204.9338154.733000000151518568.5338525.267000000

    418.2000010151517118.6677233.133000000151517045.4677117.133000000151516844.4676974000010151516474.3336838.333000000151516123.9335927.467000000151516509.36370.6000000

    419.1900000151518605.68674.933000000151518597.2678602.533000000151518919.5338787.467000000151518283.9338638000000151517699.0677639.533000000151517528.1677676.333000000

    420.1900000151516591.86382.533000000151516386.2676407.067000000151516772.2676658.133000000151515896.2676277.667000000151514923.5335029.467000000151514839.85055.333000000

    421.17000-10151519255.3339202.4000000151519321.69322.6000000151519574.89338.333000000151518593.4678817.6000000151518909.2678612.267000-1-101515180558208.2000100

    14:1LPE422.19001001515170676906.667000000151517203.27252.7330000001515175097404.333000000151516651.6676920.667000000151516909.1336621.7000000151515979.4676253.8000100

    423.2000000151518605.4678656.933000000151518482.88451.933100000151518680.1338435.2000000151517552.0677764.733000000151517682.8677552.8100000151517358.47370.067000000

    14:0LPE424.20-100-10151517441.87346.80000001515175777583.8000000151517704.87440.8000000151516962.1337006000000151516665.9336346.1330-100-10151515971.1336310.733000000

    425.2000100151518518.88497.4000000151518254.8678217000000151518203.9338033.8000000151516774.3676777.533000000151517043.4676865.533000000151516174.56139000100

    426.2100000151516781.8676519.667000000151516166.0676296.467000000151516412.5336279000000151514679.9334512.833000000151514044.1334164.067000000151513191.5333569.6000000

    427.2000000151518095.68043.6000000151517829.27746.2000000151517493.67429.4000000151515793.7676243.8000000151515132.6675020.533000000151515035.3675174.733000000

    428.2000000151515609.8675412.867000000151515192.2675297.2000000151515121.8675246.2000000131413729.9623785.571000000151412521.7332464.5000000151512769.62699.667000000

    429.08000001515110515.733105370000001515110506.26710456.8671000001515110406.210385.133000000151519735.89722000000151519744.8679603.533100000151519609.69633.667000000

    430.100000-1151518791.0678826.533000000151518679.6678678.333100000151518542.28475.467000000151517632.5677603.333000000151517896.87712.467000000151517905.27845.400000-1

    431.1700000151518738.0678709.80000001515184758444.6000000151518606.3338468.667000000151517622.47848.467000000151517412.67279.067000000151517316.8677338.2000000

    432.1900000151516028.4676073.3330000001515160485968.533000000151516089.66010.933000000151514660.74954.733000000151514473.84376.3000000151514764.44630.8000000

    433.1800000151518450.88512.867000000151517807.2677806.733000000151517876.6677761000000151517125.8677194000000151517277.5337407.067000000151516670.7336774.8000000

    434.1700000151517119.8677025.6000000151516519.7336409.667000000151516962.8677055.8000000151516837.96810.133000000151517177.5337118.333000000151515902.4675923.667000000

    18:1LPA435.1900001151518959.28981.4000000151518783.5338923.933000001151518999.89155000000151519384.7339194.333000000151518843.28918.933000000151517909.27911.067000000

    15:1LPE436.2000000151517271.6677164.333000000151516860.2677136.467000000151516978.4677264.867000000151517539.97219.867000000151517363.3337606.6100000151516294.6676561.533000000

    18:0LPA437.20-10011151519007.1339013.733000000151519273.4679276.867000000151519373.0679361.4670-100111515194749509.533000000151518612.48614.2000000151518257.9338380000000

    15:0LPE438.2100000151516825.9336678.333000000151517061.1336980.067000000151517061.27087.933000000151517126.46996.3000000151516261.0676133.167000000151515770.4675897.333000000

    439.2100000151518798.88937.333000000151518939.8678904.533000000151518827.7338774.267000000151517704.3338095.967000000151516964.26682.6000000151516554.1336737.133000000

    440.2400000151516536.4676673.867000000151517325.47303.267000000151517014.87150.933000000151516523.86890.9000000151513984.9674013.467000000151514211.5333913.667000000

    441.2100000151518547.68644.8000000151518633.0678580000000151518481.48456.2000000151516994.0677681.933000000151517085.47057.733000000151516453.0676869.067000000

    442.2400000151516262.3336234.467000000151515996.26069.4000000151516036.6676016.4000000151514359.8334808.8000000151513855.7333783.467000000151513542.63803.533000000

    443.2300000151518159.6678094.8000000151517823.0677826.133000000151517731.3337583.533000000151516382.7336827.433000000151515397.7675462.8000000151515073.0335603.133000000

    444.2400001151515456.45561.733000000151515138.6675142.933000000151515294.45150.4000000151513642.4334581.9000001151512991.1672659.833000000151512437.32783.8000000

    445.2200000151518347.2678359.4000000151517879.87851.667000000151517688.6677717.533000000151517321.2677677.733000000151517012.1336961.2000000151516551.3336692.733000000

    446.2600000151516547.4676538.667000000151516394.26435.333000000151516271.2676225.6000000151516110.0676340.033000000151515438.7335321.667000000151515539.8675570.833000000

    447.2300000151518100.9338021.2000000151518069.27964.267000000151517966.4677669.2000000151517682.27756.133000000151516721.4676476.333000000151516221.0676393000000

    448.23-1-10-1-1151515900.8675557.80-1-10-1-1151515921.8675751.467000000151515868.8675599.133000000151515510.85652000000151514585.9333909.333000000151514165.83748.5000000

    449.220000-1151518577.28624.467000000151518245.8678193.267000000151518399.9338132.13300000-1151517601.47660.8000000151517463.2677490000000151517227.1337157.333000000

    16:1LPE450.22-10-1-11151519052.8678996.4000000151519366.3339164.8670-10-1-11151519106.49015000000151519218.89096.867000000151518430.28322.1000000151517942.3338016.933000000

    451.21-100-10151518740.8678829.6000000151518528.88396.3330-100-10151518305.7338137.467000000151517688.87727.6000000151517137.6677027.533000000151516553.5336894.2000000

    16:0LPE452.23-10-1-11151519190.29066.333000000151519600.9339361.20-10-1-11151518970.6678936.333000000151519292.1339114.4000000151518385.68340.133000000151517683.57832.267000000

    453.23-11-100151519609.29618.933000000151519671.7339618.733000000151519414.7339240.2670-11-100151518864.48895.2000000151518231.28039.133000000151518133.8678354.667000000

    454.2401000151517316.4677351.267000000151517300.2677372.8000000151517022.47098.933001000151516391.0676526.433000000151515435.8675542.467000000151515835.1336112.7000000

    14:0LPG455.2300000151518702.5338740.867000000151518470.48467.4000000151518355.1338240.4000000151517478.7337370.8000000151516898.9336793.333000000151516942.4677181.8000000

    456.24-3-1-101151516560.7336277.5330-1-1-1-10151516077.66015.733100010151515859.9335949.3330-10000151514718.9334510.767000000151513809.53335980-10000151513447.0674393.467100001

    457.2200001151517881.2677889.667000000151517288.8677431000001151517182.9337091.067000000151515747.46140.733000000151515266.5675540.1000000151514584.8674810.733000000

    458.2311101151515246.06754520000001515144424795.867011101151514608.64655.667000000151513225.43950.233000000151512498.1332271.667000000151512065.1332155.233000000

    459.25101001515110129.610353.333010000151519743.89617.933000000151519211.49453.867000000151519709.1339902.133000000151517523.8677576.933000000151516480.26755.2000100

    460.2620000151518561.2678912.6010000151518019.67836.6670000001515171337669000000151518139.1338406.667010000151515418.8335546.067000000151514068.24544.267000000

    461.2410000151518397.48561.867010000151517845.8677805000000151517666.8677637.133000000151517649.27644.133000000151516744.1336628000000151515588.2675830000000

    462.2400001151516906.6676900.067000000151516314.66471.267000001151516162.8676213.333000000151516716.7336592.6000000151516413.4676636.133000000151515390.6675039.533000000

    463.2300000151517740.27739.533000000151517661.6677470.733000000151517434.6677318.4000000151516876.9337090000000151516582.0676612000000151516421.1336283000000

    17:1LPE464.2400000151516908.5336814.067000000151516880.8676810.533000000151516500.1336562.667000000151516959.86667.167000000151516401.9336466000000151515851.35863.933000000

    465.2400000151519004.8678942.6670000001515191719184.133000000151519176.2679042.867000000151518838.0678781.067000000151518457.88364.8000000151518398.28487.4000000

    17:0LPE466.2500000151517157.7337049.733000000151517430.9337556.2000000151517422.87405.333000000151517510.6677357.533000000151516846.7336865.2000000151516940.8676922.533000000

    467.2300000151518119.0678110.333000000151518102.9338119.667000000151517941.4677851.533000000151517145.77109.733000000151516661.9336654.733000000151516608.5336625.133000000

    468.240-1001151516060.9335863.267000000151516410.9336384.667000000151516069.0676088.13300-10011515158726010.233000000151514547.14249.533000000151514367.6674103.433000000

    469.230000-1151517905.1337907000000151517629.2677648.867000000151517734.87601.2000000151516327.2336789.4000000151516249.26064000000151515936.65720.46700000-1

    470.2300000151515563.25423.667000000151515108.3335358.867000000151515245.9335033.8000000151513685.1674516.8000000151513507.7333405.933000000151513559.33608.15000000

    471.23000-10151518143.6678221.067000000151518132.9338027.867000000151518146.67912.267000000151516506.5336678.733000000151516179.8675718.933000000151515987.6676270.2671000-10

    472.2600000151515973.65942.867000000151515972.2675896.533000000151516019.6675981.533000000151515119.6675261.6000000151514459.5334092.967000000151514388.7334779.533000000

    473.2400000151519161.6679311.267000000151518723.6678650.333000000151518762.68713.0670000001515183638532.933000000151518110.5338008.533000000151517692.87733.533000000

    18:3LPE474.2400000151517564.4677627.667000000151517391.8677318.6000000151517139.0677073.2000000151517206.0337205.067000000151516620.0676873.467000000151516206.3336331.267000000

    475.2400000151517500.5337579.267000000151517082.8677032.933000000151517152.3337057.6000000151515965.0336297.067000000151515697.7335683.733000000151515825.25633.133000000

    18:2LPE476.21-2-1-1-1-11515181718087.8670-1-1000151518921.0678666.60-10-1-10151518262.68259000000151518632.3678542.93310000-1151517557.5337514.467000000151517263.6677345.8100000

    477.241-1111151518003.9338014000000151517961.2677774.467000000151517623.67596.2000000151517434.9337336.1000000151516819.0676806.067000000151516372.2336512.73301-1111

    18:1LPE478.22-1-1-1-111515110909.610886.73300-1000151511112411021.5330-10-1-111515110995.411004.3330000001515111153.73311125.6670000001515110708.86710719.5330000001515110243.06710315.6100000

    479.23-10-2-11151519887.49860.1330000001515110254.93310044.1330-10-1-111515110012.5339972.667000-1001515110195.13310091.067000000151519440.4679414.5330000001515188618894.867100000

    18:0LPE480.24000-10151519054.28930.2000000151519394.49244.20000-10151519114.6679157.333000000151519861.7339669.6000000151519287.89233.8670000001515183468318.267000000

    16:1LPG481.2200000151518803.4678796.067000000151518760.3338800.267100000151519063.2679060.2000000151519143.0678991.8000000151518649.8678592.667000000151518127.48102.067000000

    482.2300000151516183.66245000000151516272.1336418.467000000151516624.8676608.267000000151516640.0336261.733000000151516000.26033.4000000151515826.55881.933000000

    16:0LPG, 26:0DAG483.2100000151518525.6678422000000151518517.28465.81000001515188858979.2000000151518846.88644.467000000151518410.7338362.133000000151517977.0677986.267000000

    484.2300000151515743.8675871.4000000151515752.4675883.333100000151516313.86320000000151516286.4675970.6000000151515965.8335892.933000000151515238.3335697.933000000

    485.22021101515172597266.133000001151517113.66998.533000000151517006.0676970.333000000151515714.0675628.4000000151516062.2676185.9331010001515155565780.46700111-1

    486.24000-10151515120.7334947.7330000-10151514742.3334734.267000000151514692.7334656.6000000151513630.83542.133000000151513481.0333485.4000000151512899.8672995.8000000

    487.2100000151516795.4676801.067000000151516142.2676224.933000000151516209.4676262.467000000151514172.6334374.4000000151514332.3333830.467000000151513724.23785.333000000

    13:0LPC(Cl)488.35-10000151517835.3337847000000151517952.3337669.533000000151516950.8677039.6000000151516993.3336849.033000000151517375.0677409.9330-10000151516320.6336201000000

    489.27-10000151518698.0678706.267000000151517906.47870.6100000151517747.47668.933000000151516998.2336967.767000000151517541.5337575.6670-10000151516735.4676908.067000000

    490.2600000151517064.67046.133000000151517031.3336951.533000000151516725.5336740.867000000151516643.7336591.1000000151516250.16356.133100000151515859.8675747.067000000

    491.26-100-10151517647.87606.8671000-10151516860.6676819.667000000151516565.3336575.733000000151516152.66421.567000000151515762.3335657.6670-10000151515436.7335295.8000000

    19:1LPE492.25000-10151516790.9336628.3330000001515168076586.20000-101515164156451.3330000001515169266641.167000000151516405.6676521.467000000151515516.8675136.617000000

    493.25-10000151517069.8677114.133000000151516681.46641.2000000151516472.66438.667000000151515822.8676012.567000000151515745.1335656.8670-10000151515101.2675348.933000000

    16:1LPS, 19:0LPE494.2400000151517127.66833000000151516560.6676555000000151516607.46625.667000000151517038.6336761.1000000151517006.4677039.733000000151516059.85875.2000000

    495.2510010151519234.29490010000151519305.3339236.467100010151519254.9339334.933100-100151518672.5339093.3000000151516880.0336746.533000000151516190.3336674000100

    16:0LPS496.2520010151517797.5338046.401-1100151517825.5337785.467100010151517804.2677933.4011-100151517451.17729.4000000151516135.0676323.2000000151515094.0335361.533000000

    497.2520110151518195.5338420.26701-1100151518178.9338191100010151518129.68274.467011000151517245.477780000001515157615325.467000000151514978.3335308.733000000

    20:5LPE498.220-1-100151518483.0678561.06700-1000151519158.3338922.2100-100151518753.88814.2000000151518780.287290000001515176627603.867000000151517453.1337667.333100000

    499.23-10000151517374.47372.8000000151517595.3337463.1330000001515172507256.8000000151516782.5336659.233000000151515803.53357260-10000151515715.5335535.833000000

    20:4LPE, 14:1LPC(Cl)500.22-10-2-111515110504.73310498.40000001515110965.66710800.2670-10-1-111515110563.06710524.6000-1001515110741.33310640.333000000151519903.69877.333100000151519656.69791.933100000

    501.2300-200151519141.89132.2000000151519861.2679581.067100-100151519087.5339024.067000000151519192.0679040.2000000151518266.6678150.133100-100151518053.9338089100000

    20:3LPE, 14:0LPC(Cl)502.23-1-1-2-10151519938.6679895.3330000001515110380.53310133.5330-1-1-1-101515110096.66710046.667000-100151511019610092.467000000151519357.8679345.133000000151518920.29034.533100000

    503.25-100-20151518239.5338223.667000000151518594.68367.6670-100-10151518215.48254.9330000001515182368211.8000000151517481.2677586.6671000-10151517086.87294.733100000

    20:2LPE504.27-10-2-101515185708525.2000000151519022.2678732.60-10-1-10151518727.9338845.667000000151518937.28826.533000000151518224.9338178.4100-100151517674.5337749.8100000

    505.2600000151518102.3338083.867000000151517774.3337748.8000000151517458.6677501.2000000151517308.47199100000151517613.9337770.467000000151516947.7336828.133000000

    20:1LPE506.28-1-1-1-11151519667.89606.2670000001515110085.0679875.4670-1-1-1-111515110053.810136.8670000001515110383.210255.8000000151519954.1339942.8000000151519071.4679031.8000000

    18:2LPG507.27000-11151518623.1338528.067000000151518661.5338596.9330000-11151518833.0678949.133000000151518944.68825.80000001515186238605.40000001515179777894.533100000

    20:0LPE508.31000-1-1151517846.3337713.667000000151517885.8677743.5330000-10151517702.6677879.667000000151518120.1337768.33300000-1151517911.87949.6000000151517628.5337431.733000000

    18:1LPG509.24-10-1-1-115151100799924.3330-10-1-1-11515110170.73310161.7331000001515110564.73310653.8670000001515110287.06710170.7330000001515110248102010000001515110020.410017.933000000

    510.24000001515181457931.6000000151518295.9338313.41000001515189379026.533000000151518456.9338332.667000000151518554.88488.1000000151518300.4678321.667000000

    18:0LPG, 28:0DAG511.2500001151517848.8677685.2000000151517593.9337760.7330000011515181048181.067000000151517221.47271.667000000151517633.2677592.033000000151517543.4677610.733000000

    512.26-1-1-1-10151515529.2675175.2670-1-1-1-10151516086.26147.467000000151515830.66166.667000000151515681.5335500.9000000151515280.84995.2000000151515314.7335399.067100000

    513.2600100151516399.4676336.733000000151516169.2676211.6670000001515159555935.40000001515143834160.467000000151514344.4674371.767000000151513897.0674505.667000100

    15:1LPC(Cl)514.25010-10151515889.45566.867000000151516536.66425.533000000151515786.46074.4000000151516563.5336260.90000001515153195176.133000000151514665.5334882.5331010-10

    515.2700000151516251.0676165000000151515970.3335984.667000000151515440.3335694.4000000151514688.1335230.933000000151513705.63402.8000000151513581.5673551.133000000

    15:0LPC(Cl)516.29000-1-1151516816.7336776.667000000151516977.0676766.6670000-10151516363.86612.267000000151516879.2676587.33300000-1151516234.1336077.367000000151515420.5335430.233000000

    517.2800000151515943.4675959.333000000151515776.5335704.533000000151515184.0675318.8000000151514533.8674822.2000000151514441.4674192.967000000151514172.9334118.5000000

    518.41000-10151519874.1339895.733000000151519497.2679315.0671000-10151518112.28354.467100000151517700.67585.6000000151518865.5339056.133000000151518231.27915.8000000

    519.39000-10151518773.6678863.867000000151518093.4677922.7331000-10151516773.1337034.533100000151516217.1676072.833000000151517440.8677626.8000000151516792.7336541.667000000

    18:2LPS520.34000-10151517789.5337677.933000000151517235.27051.1331000-10151516310.46615000000151516784.46443.5000000151517312.87522.533000000151516209.5675781.533000000

    521.3100100151516629.2676508.467000000151515785.7335802000000151515434.0675544.4000000151514591.9334214.4000000151515247.1675655.267100100151514618.1334171.967000000

    18:1LPS522.300-1-1-20151519228.49152.667000000151519000.33388130000-101515189169084.533000000151519158.88815.267000000151518992.1339041.733000000151518460.7338267.46700-1-1-10

    523.2900-100151517732.3337631.067000000151517307.8677224.533100000151517297.8677374.6000000151517171.1336822.567000000151517193.6677268.2000000151516804.9336480000-100

    18:0LPS, 22:6LPE524.25-10-1-111515110190.46710178.0670000001515110533.46710335.5330-10-1-111515110197.86710217.80000001515110363.26710262.067000000151519671.89695.467000000151519192.7339211.067100000

    525.24-10-1-11151518697.88716.333000000151519075.7338895.1330-10-1-11151518518.48542.733000000151518674.3338481.467000000151517909.0677966.067000000151517537.7337514.8100000

    22:5LPE526.24-10-1-10151519878.0679854.2670000001515110498.93310291.4670-10-1-10151519893.5339908.0670000001515110076.5339928.333000000151519168.89115.533100000151518851.2679041100000

    527.25-3-2-1-1-1151518429.06783230-1-1100151519004.8678813.20-10-1-10151518227.28135.933000000151518427.1338024.20-1-1-10-1151517491.1337458.867100000151517205.47301.667100000

    22:4LPE, 16:1LPC(Cl)528.25-10-1-11151519089.4679020.667000000151519833.29595.4670-10-1-11151519147.9339182.6000000151519336.7339241.0330000001515187218737.933000000151518135.28236100000

    529.250-10-10151517528.8677419.73300-1000151518089.279560000-10151517459.87541.333000000151517410.9337057.3000000151516994.27146.667000000151516232.2676516.667100000

    22:3LPE, 16:0LPC(Cl)530.26-10-202151519615.69551.4000000151519928.29702.80-10-1-11151519804.9339759.4000-111151519840.5339794.467000000151519260.1339271.6000000151518545.68621.8100000

    20:4LPG531.26000-10151517803.6677857.4000000151518097.6677922.20000-10151518027.7338007.933000000151518089.9337770.8000000151517535.47557.333000000151516845.86916.933100000

    22:2LPE532.2800-1-11151517879.3337863000000151518214.6678009.133000-1-11151518006.3338176.333000000151518393.2678275.067000000151517812.67821.867000000151516919.1337101.533000000

    20:3LPG533.2700000151516728.3336729.133000000151516493.4676569.3330000001515169126884.467000000151516383.9336447.8000000151516414.5336445.867000000151515948.25874.8000000

    22:1LPE534.34-100-10151518387.6678393.133000000151518625.8678411.4670000-10151518313.9338414.933000000151518553.88376.2670000001515181898189.8670-10000151517895.0677867.533000000

    20:2LPG535.32000-10151517618.8677712.2670000001515175217350.2670000-10151517326.1337519.067000000151517154.4676946.3000000151517220.87222.267000000151517005.4677036.067000000

    C16:0S536.431-1-1-101515111201.13311210.467000000151511110310972.933100000151511094410945.4671000001515110804.73310753.40000001515111074.611115.61000001515111007.7331082201-1-1-10

    20:1LPG537.431-1-1-101515110625.73310639.5330000001515110442.86710228.61000001515110143.13310168.667100000151519795.1339715.60000001515110326.410406.66710000015151102729982.33301-1-1-10

    538.421-1-1-20151519609.0679599000000151519418.89122.3330000-10151518989.28941.6000000151518726.0678608.733000000151519212.49280.133100000151519160.88843.06701-1-1-10

    30:0DAG539.381-1-1-2-1151518021.0678119.333000000151517557.4677390.9330000-10151516976.3336908.533000000151516691.8676303.5330000-1-1151517219.3337403.333100000151517256.4676986.33301-1-100

    540.3500000151516090.7336032.9330000001515158895974.667000000151515448.25535.2000000151515413.9335561.667000000151515336.85211.067000000151514807.6674863.067000000

    541.