dual parallel mass spectrometers with 31 p nmr for sphingolipid analysis wm. craig byrdwell a...
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Dual Parallel Mass Spectrometers with 31P NMR for Sphingolipid Analysis
Dual Parallel Mass Spectrometers with 31P NMR for Sphingolipid Analysis
Wm. Craig Byrdwella
Richard H. Perryb
aFood Composition Laboratory, USDA, ARS,
Beltsville Human Nutrition Research Center
bPurdue UniversityDept. of Chemistry
Wm. Craig Byrdwella
Richard H. Perryb
aFood Composition Laboratory, USDA, ARS,
Beltsville Human Nutrition Research Center
bPurdue UniversityDept. of Chemistry
OverviewOverview
• What are Sphingolipids?• Why study Sphingolipids?• What information can APCI-MS provide?• What information can ESI-MS provide?• Why Dual Parallel Mass Spectrometers?• Why use 31P-NMR? What information?• What about quantification?
• What are Sphingolipids?• Why study Sphingolipids?• What information can APCI-MS provide?• What information can ESI-MS provide?• Why Dual Parallel Mass Spectrometers?• Why use 31P-NMR? What information?• What about quantification?
H2N
OH
OH
H
H
SCoA
O
H2N
H2C
OH
OH
H
O
Serine
Palmitoyl-CoA
+
NADPH + H+
3-Ketosphinganine reductase
NADPCO2
Serine palmitoyltransferase
What are Sphingolipids?What are Sphingolipids?
H2N
OH
O
H
They are a FAMILY of molecules based on the ceramide backbone
3-KetosphinganineSphinganine
, formed as follows :
O
H2N
OH
OH
H
H
CoASH
Fatty Acyl CoA
Ceramide synthase
NH
OH
O
OH
H
H
DesaturasePhosphocholine transferase
transfers choline from GPCho
NH
O
O
OH
PO
H2C
CH2
N+ CH3
O
OH
CH3
CH3H
H
Sphinganine
NH
OH
O
OH
H
H
DihydroceramideCeramideSphingomyelin !
What are Sphingolipids?What are Sphingolipids?
The Sphingolipids we’re interested in:The Sphingolipids we’re interested in:
NH
O
O
OH
PO
H2C
CH2
N+ CH3
O
OH
CH3
CH3H
H
NH
O
O
OH
PO
H2C
CH2
N+ CH3
O
OH
CH3
CH3H
H
Sphingomyelin (d18:1/18:1 SM)
Dihydrosphingomyelin (d18:0/18:1 DSM)
Why Study Sphingomyelin?Why Study Sphingomyelin?
• Sphingomyelins are major components in the plasma membrane bilayers of cells.
• Sphingomyelins have special properties:– They self-associate into ‘membrane rafts’– They have a higher gel-to-liquid crystalline phase
transition temperature than glycerophospholipids• They play crucial roles in cellular signaling:
– Sphingomyelinase acts on sphingomyelin to produce ceramide, a potent cell signaling molecule
• They have been mischaracterized.
• Sphingomyelins are major components in the plasma membrane bilayers of cells.
• Sphingomyelins have special properties:– They self-associate into ‘membrane rafts’– They have a higher gel-to-liquid crystalline phase
transition temperature than glycerophospholipids• They play crucial roles in cellular signaling:
– Sphingomyelinase acts on sphingomyelin to produce ceramide, a potent cell signaling molecule
• They have been mischaracterized.
Results in the past have been mixed…
With some agreement,
But Substantial disagreement
Results in the past have been mixed…
With some agreement,
But Substantial disagreement
Why Study Sphingomyelin?Why Study Sphingomyelin?Author Sample Technique
SM Species
DSM Species
Kerwin et al.
(1994)Bovine Brain
ESI-MS
(infusion)15 2
Han & Gross
(1995)Bovine Brain
ESI-MS
(infusion)6 0
Hsu & Turk
(1999)
Bovine Brain
B.Erythrocytes
Chicken Egg
ESI-MS (Li+)
(infusion)
19
17
12
0
1
0Karlsson et al.
(1998)
Bovine Brain
Bovine MilkESI-MS
15
23
1
5
Byrdwell & Borchman (1997)
Chicken Egg
Human Lens
Human Lens
APCI-MS
APCI-MS
ESI-MS
12
11
12
6
13
14Byrdwell
(1998)
Bovine Brain
Pig LensESI-MS
23
19
19
15
APCI-MS, MS/MS and MS3
OfBovine Brain Sphingolipids
APCI-MS, MS/MS and MS3
OfBovine Brain Sphingolipids
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
RT: 0.00 - 60.14
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min)
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
nd
an
ce
34.25
30.75
35.24
35.50
35.65
36.33
27.2326.87
NL:1.35E10
TIC MS BBS_+ve APCI ms3a_1
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
RT: 20.00 - 45.00
20 22 24 26 28 30 32 34 36 38 40 42 44
Time (min)
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
nd
an
ce
34.25
34.10
30.7531.0230.55
31.16
31.36
32.70
35.2435.34
35.65
29.96
29.70
36.33
27.2326.8727.59
26.40
38.86
NL:1.35E10
TIC F: + c APCI Full ms [ 200.00-1200.00] MS BBS_+ve APCI ms3a_1
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
RT: 22.06 - 42.00
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
RT: 34.25
RT: 30.75
RT: 27.23
NL: 1.35E10
TIC F: + c APCI Full ms [ 200.00-1200.00] MS ICIS BBS_+ve APCI ms3a_1
BBS_+ve APCI ms3a_1 #1039-1269 RT: 25.00-28.96 AV: 77 NL: 2.89E8T: + c APCI Full ms [ 200.00-1200.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
632.7
606.8
634.7
578.7635.7
660.6284.4266.4 576.8
See masses at www.Sphingomyelin.com
22:0 DSM
20:0 DSM
NH2+
O
OHH
NH2+
O
OHH
NH2+
O
OHH
NH2+
O
OHH
24:0 DSM
24:1 DSM
*
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
RT: 22.06 - 42.00
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
RT: 34.25
RT: 30.75
RT: 27.23
NL: 1.35E10
TIC F: + c APCI Full ms [ 200.00-1200.00] MS ICIS BBS_+ve APCI ms3a_1
BBS_+ve APCI ms3a_1 #1269-1503 RT: 29.02-33.46 AV: 78 NL: 1.49E9T: + c APCI Full ms [ 200.00-1200.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
632.6
604.7
630.6
576.6
550.8 633.6264.5
659.5282.4 736.3
See masses at www.Sphingomyelin.com
22:0 SM
20:0 SM
24:0 SM
24:1 SM
NH2+
O
OHH
NH2+
O
OHH
NH2+
O
OHH
NH2+
O
OHH
NH2+
O
OHH
18:0 DSM*
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
RT: 22.06 - 42.00
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
RT: 34.25
RT: 30.75
RT: 27.23
NL: 1.35E10
TIC F: + c APCI Full ms [ 200.00-1200.00] MS ICIS BBS_+ve APCI ms3a_1
BBS_+ve APCI ms3a_1 #1503-1818 RT: 33.51-38.97 AV: 105 NL: 2.11E9T: + c APCI Full ms [ 200.00-1200.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
548.5
530.8576.5
264.5 520.7 654.3282.4 717.3
See masses at www.Sphingomyelin.com
20:0 SM
18:0 SM
16:0 DSM
[(Cer+H)-2H2O]+
NH2+
O
OHH
NH2+
O
OHH
NH2+
O
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
BBS_+ve APCI ms3a_1 #1039-1269 RT: 25.00-28.96 AV: 77 NL: 2.89E8T: + c APCI Full ms [ 200.00-1200.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
632.7
606.8
634.7
578.7635.7
660.6284.4266.4 576.8
BBS_+ve APCI ms3a_1 #1039-1269 RT: 26.31-28.68 AV: 17 NL: 2.02E7F: + c d Full ms2 632.70
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
614.6
284.2266.2 615.6390.4 613.6366.3331.3
APCI-MS, MS/MS and MS3
of
Bovine Brain Dihydrosphingomyelin
See masses at www.Sphingomyelin.com
[LCB-H2O]+
24:1 DSM
[LCB]+
[Cer-H2O+H]+
[Cer-2H2O+H]+
[24:1 FA(L)]+
[24:1 FA(S)]+
+H3N
OHHNH2+
O+H3N
NH+
O
NH3+
O
NH2+
O
OHH
SL1
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
BBS_+ve APCI ms3a_1 #1039-1269 RT: 26.57-28.68 AV: 15 NL: 2.29E7F: + c d Full ms2 632.60
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
614.6
284.2266.2 615.6390.4 613.6366.3331.3
BBS_+ve APCI ms3a_1 #1039-1269 RT: 26.32-28.70 AV: 17 NL: 2.04E5T: + c d Full ms3 632.65@54.00 614.62@54.00 [ 155.00-620.00]
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
586.7 596.6
360.3
266.2320.4
530.7 558.5516.6254.1 488.6
404.4390.3 446.4 502.5432.5376.2 572.6474.3 544.5348.4
306.4 597.9418.5184.1 304.2 346.4191.7 221.3
See masses at www.Sphingomyelin.com
[LCB-H2O]+
[Cer-2H2O+H]+
[Cer-3H2O+H]+
[24:1 FA(L)]+
NH2+
O
14 = CH2
*
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
BBS_+ve APCI ms3a_1 #1269-1503 RT: 29.02-33.46 AV: 52 NL: 1.49E9F: + c APCI Full ms [ 200.00-1200.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
632.6
630.6
576.6
550.8 633.6264.5
659.5282.4 736.3
BBS_+ve APCI ms3a_1 #1269-1503 RT: 29.14-30.88 AV: 13 NL: 7.50E7F: + c d Full ms2 632.70
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
586.7
614.5
613.6
392.5264.3
282.2 391.4 427.5368.3
APCI-MS, MS/MS and MS3
of
Bovine Brain Sphingomyelin
See masses at www.Sphingomyelin.com
[LCB-H2O]+
24:0 SM
[LCB]+
[Cer-H2O+H]+
[Cer-2H2O+H]+
[24:0 FA(L)]+
[24:0 FA(S)]+
+H3N
OHHNH2+
O+H3N
NH+
O
NH3+
O
NH2+
O
OHH
SL2
BBS_+ve APCI ms3a_1 1/26/2003 12:43:59 PMBBS (10.87mg/mL), +ve APCI-MS,Tray Temp = 32 Deg., Oven Temp=57 Deg.,10uL inj., 80% Hex:20%H2O Iso.,CH430=57 Deg.,Alltech-Alltech,PDA,APCI
BBS_+ve APCI ms3a_1 #1269-1503 RT: 29.14-30.88 AV: 13 NL: 7.50E7F: + c d Full ms2 632.60
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
614.5
613.6
392.5264.3
282.2 391.4 602.4427.5368.3
BBS_+ve APCI ms3a_1 #1269-1503 RT: 29.16-30.74 AV: 11 NL: 9.41E6T: + c d Full ms3 632.59@54.00 614.49@54.00 [ 155.00-620.00]
200 250 300 350 400 450 500 550 600
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
392.5
264.2 427.2391.2 529.3403.3 427.9361.5 595.5516.6460.3262.1223.2 265.4192.0
See masses at www.Sphingomyelin.com
[LCB-H2O]+
[Cer-2H2O+H]+
[24:0 FA(L)]+
NH2+
O
ESI-MS and APCI-MS Simultaneously
(Dual Parallel MS)
Of
Bovine Brain Sphingolipids
ESI-MS and APCI-MS Simultaneously
(Dual Parallel MS)
Of
Bovine Brain Sphingolipids
bbs +ve apci dual quan_c 1/28/2004 9:21:53 PM
RT: 0.00 - 59.99 SM: 7G
0 10 20 30 40 50Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.84
31.87
NL:1.08E9
TIC MS bbs +ve apci dual quan_c
RT: 0.00 - 60.00 SM: 7G
0 10 20 30 40 50 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.73
26.06 36.3834.77
32.60 37.66
NL:7.10E6
TIC MS 0128bbs3
bbs +ve apci dual quan_c #1152-1353 RT: 26.01-29.99 AV: 202 NL: 2.38E7T: + c APCI Full ms [ 200.00-1200.00]
200 300 400 500 600 700 800 900m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
632.6
606.7634.6
635.6551.5
266.4
284.4 660.6313.3 756.4264.4 390.5 550.7 837.4734.4 783.4
0128bbs3 #1032-1198 RT: 24.99-29.00 AV: 167 NL: 1.33E6T: + c ESI Full ms [ 150.00-2000.02]
200 300 400 500 600 700 800 900m/z
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
734.4815.5
817.6
789.7
756.4818.5
423.3839.2221.2
391.1 706.5424.3 845.5230.9 679.5274.4
x6.7
C:\Documents and Settings\...\0128bbs3 1/28/2004 9:23:40 PM BBS (10.22mg/mL)+ DPPC (0.500035mg/mL)2 TEES: UV/ELSD, LCQ-APCI*TSQ-ESI, 20mM NH4OCOH@20uL/min Sheath
RT: 24.00 - 42.00 SM: 7G
24 26 28 30 32 34 36 38 40 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.8435.47
36.0131.8732.05
31.5632.49
36.34
36.6333.00
NL:1.08E9
TIC MS bbs +ve apci dual quan_c
RT: 24.00 - 42.00 SM: 7G
24 26 28 30 32 34 36 38 40 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.7336.02
26.06 36.3834.7726.5136.7937.0832.6025.67 26.78 31.87
31.3227.21 30.81
NL:7.10E6
TIC MS 0128bbs3
bbs +ve apci dual quan_c #1152-1353 RT: 26.01-29.99 AV: 202 NL: 2.38E7T: + c APCI Full ms [ 200.00-1200.00]
500 520 540 560 580 600 620 640 660 680 700m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
632.6
606.7634.6
614.7
635.6551.5
578.7 588.8 616.7604.7 620.7
552.5 660.6589.8636.6550.7 561.7 662.6
0128bbs3 #1074-1198 RT: 26.01-29.00 AV: 125 NL: 1.25E6T: + c ESI Full ms [ 150.00-2000.02]
700 720 740 760 780 800 820 840 860 880 900m/z
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
734.4
815.6
817.5
789.7736.5
818.5790.6789.2
756.4
803.5787.5761.5 837.4
737.6 843.7762.6706.5 829.4720.4 755.3
x4.5
APCI-MSAPCI-MS ESI-MSESI-MS
[PP]+
24:0 DSM
22:0 DSM
20:0 DSM
-H2O
-H2O
24:1 DSM DPPC 24:1
DSM
24:0 DSM
22:0 DSM
20:0 DSM
-CH2
+Na+Na
C:\Documents and Settings\...\0128bbs3 1/28/2004 9:23:40 PM BBS (10.22mg/mL)+ DPPC (0.500035mg/mL)2 TEES: UV/ELSD, LCQ-APCI*TSQ-ESI, 20mM NH4OCOH@20uL/min Sheath
RT: 24.00 - 42.00 SM: 7G
24 26 28 30 32 34 36 38 40 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.8435.47
36.0131.8732.05
31.5632.49
36.34
36.6333.00
NL:1.08E9
TIC MS bbs +ve apci dual quan_c
RT: 24.00 - 42.00 SM: 7G
24 26 28 30 32 34 36 38 40 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.7336.02
26.06 36.3834.7726.5136.7937.0832.6025.67 26.78 31.87
31.3227.21 30.81
NL:7.10E6
TIC MS 0128bbs3
bbs +ve apci dual quan_c #1353-1584 RT: 29.99-34.50 AV: 232 NL: 6.46E7T: + c APCI Full ms [ 200.00-1200.00]
500 520 540 560 580 600 620 640 660 680 700m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
612.6
630.5
613.6
614.6
631.5550.6 586.6558.6 604.6576.5
600.7633.5559.6532.7
658.5560.6
0128bbs3 #1239-1426 RT: 29.99-34.50 AV: 188 NL: 7.65E5T: + c ESI Full ms [ 150.00-2000.02]
660 680 700 720 740 760 780 800 820 840m/z
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
813.5
759.5 814.5
787.5 815.6
733.5
760.5788.5734.4 816.5
761.5 801.5841.5817.5735.4 762.5731.5705.5 843.4
APCI-MSAPCI-MS ESI-MSESI-MS
18:0DSM
24:0 SM22:0
SM20:0 SM
-H2O
-H2O
24:1 SM
18:0DSM
24:1 SM
24:0 SM22:0
SM
20:0 SM
16:0DSM
-H2O
C:\Documents and Settings\...\0128bbs3 1/28/2004 9:23:40 PM BBS (10.22mg/mL)+ DPPC (0.500035mg/mL)2 TEES: UV/ELSD, LCQ-APCI*TSQ-ESI, 20mM NH4OCOH@20uL/min Sheath
RT: 24.00 - 42.00 SM: 7G
24 26 28 30 32 34 36 38 40 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
35.8435.47
36.0131.8732.05
31.5632.49
36.34
36.6333.00
NL:1.08E9
TIC MS bbs +ve apci dual quan_c
RT: 24.00 - 42.00 SM: 7G
24 26 28 30 32 34 36 38 40 42Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
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35.7336.02
26.06 36.3834.7726.5136.7937.0832.6025.67 26.78 31.87
31.3227.21 30.81
NL:7.10E6
TIC MS 0128bbs3
bbs +ve apci dual quan_c #1584-1814 RT: 34.50-39.00 AV: 231 NL: 1.17E8T: + c APCI Full ms [ 200.00-1200.00]
500 520 540 560 580 600 620 640 660 680 700m/z
0
10
20
30
40
50
60
70
80
90
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Rel
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bund
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548.4
530.6
549.4
531.6
558.5 576.4544.6
559.5 577.4520.5 612.5
0128bbs3 #1426-1613 RT: 34.50-39.01 AV: 188 NL: 2.22E6T: + c ESI Full ms [ 150.00-2000.02]
660 680 700 720 740 760 780 800 820 840m/z
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
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731.4
732.4
733.5 759.4
760.4703.4 753.4 813.5787.3
APCI-MSAPCI-MS ESI-MSESI-MS
18:0 SM
16:0 SM
-H2O
-H2O
20:0 SM
20:0 SM
18:0 SM
16:0 SM +Na
-H2O
C:\Documents and Settings\...\0128bbs3 1/28/2004 9:23:40 PM BBS (10.22mg/mL)+ DPPC (0.500035mg/mL)2 TEES: UV/ELSD, LCQ-APCI*TSQ-ESI, 20mM NH4OCOH@20uL/min Sheath
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10027.07 27.32 NL: 3.24E7
m/z= 634.1-635.1 MS bbs +ve apci dual quan_c
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10027.50
27.1227.79
NL:4.72E5m/z= 817.2-818.2 MS 0128bbs3
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10027.8227.56
31.1828.54 31.68
NL: 5.21E7
m/z= 632.1-633.1 MS bbs +ve apci dual quan_c
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10030.78
27.65 31.4427.8227.31 32.07
NL:7.93E5m/z= 815.2-816.2 MS 0128bbs3
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10029.60
30.01NL: 1.78E7
m/z= 578.0-579.0 MS bbs +ve apci dual quan_c
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10029.6529.15
29.96NL:4.72E5m/z= 761.1-762.1 MS 0128bbs3
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10034.05
33.78 34.52NL: 6.42E7
m/z= 576.0-577.0 MS bbs +ve apci dual quan_c
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10034.16
34.4133.56
NL:1.70E6m/z= 759.1-760.1 MS 0128bbs3
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10031.02
31.14NL: 4.85E7
m/z= 550.0-551.0 MS bbs +ve apci dual quan_c
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10030.88
30.62 31.12 35.95
NL:9.20E5m/z= 733.1-734.1 MS 0128bbs3
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10035.84 NL: 2.62E8
m/z= 548.0-549.0 MS bbs +ve apci dual quan_c
24 26 28 30 32 34 36 38 40 42Time (min)
0
50
10035.9535.73 36.26
35.06 37.68
NL:4.13E6m/z= 731.1-732.1 MS 0128bbs3
APCI-MSAPCI-MS ESI-MSESI-MS24:0 DSM 24:0 DSMm/z 634.6 m/z 817.7
24:1 DSM 24:1 DSMm/z 632.6 m/z 815.7
20:0 DSM 20:0 DSMm/z 578.5 m/z 761.6
20:0 SM 20:0 SMm/z 576.5 m/z 759.6
18:0 DSM 18:0 DSMm/z 522.5 m/z 733.6
18:0 SM 18:0 SMm/z 548.5 m/z 731.6
24:0 SM 24:0 SM
31P Nuclear Magnetic Resonance (NMR) Spectroscopy
OfSphingolipids
31P Nuclear Magnetic Resonance (NMR) Spectroscopy
OfSphingolipids
31P NMR Spectroscopy31P NMR Spectroscopy
-0.500.00
DPPC (I.S.)
-0.840
DPPC (I.S.)
-0.840
SM-0.081
SM-0.081
DSM+0.133DSM
+0.133
-0.500.00
DPPC (I.S.)
-0.840
DPPC (I.S.)
-0.840
SM-0.081
SM-0.081
DSM+0.133DSM
+0.133
1.0143 6.9094 3.0463
DPPC DSM SM
3.0463 6.9094 1.0143
12.8% 87.2%
11.4(g/mL)
3.82(g/mL)
25.93(g/mL)
31P NMR Spectroscopy31P NMR Spectroscopy
-0.500.00
DPPC (I.S.)
-0.840
DPPC (I.S.)
-0.840
SM-0.084
SM-0.084
DSM+0.129DSM
+0.129
31P NMR Spectroscopy of Bovine Milk
31P NMR Spectroscopy of Bovine Milk
-0.500.00
DPPC (I.S.)
-0.840
DPPC (I.S.)
-0.840
SM-0.084
SM-0.084
DSM+0.129DSM
+0.129
0.9853 5.1273 2.3353
DPPC DSM SM
2.3353 5.1273 0.9853
16.1% 83.9%
10.6(g/mL)
4.47(g/mL)
23.27(g/mL)
31P NMR Spectroscopy of Bovine Milk
31P NMR Spectroscopy of Bovine Milk
Dual Parallel Mass Spectrometry(APCI-MS & ESI-MS)
ofBovine Milk Sphingolipids
Dual Parallel Mass Spectrometry(APCI-MS & ESI-MS)
ofBovine Milk Sphingolipids
c:\documents and settings\...\0128bms2 1/28/2004 3:09:24 PM BMS (10.58mg/mL)+ DPPC (0.500035mg/mL)2 TEES: UV/ELSD, LCQ-APCI*TSQ-ESI, 20mM NH4OCOH@20uL/min Sheath
RT: 0.00 - 59.98
0 10 20 30 40 50Time (min)
0
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20
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40
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60
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Rel
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32.46
32.01
28.98 37.2538.60
40.27
NL:1.15E9
TIC MS BMS +ve APCI Dual Quan_b
RT: 0.00 - 60.01 SM: 5G
0 10 20 30 40 50 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
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bund
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32.78
32.32 33.62
26.2134.03
37.15 37.53
38.50
39.80
40.79
41.37
NL:8.53E6
TIC MS 0128bms2
RT: 25.00 - 45.00
26 28 30 32 34 36 38 40 42 44Time (min)
0
10
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30
40
50
60
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Rel
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NL:1.15E9
TIC MS BMS +ve APCI Dual Quan_b
RT: 25.00 - 45.00 SM: 5G
26 28 30 32 34 36 38 40 42 44Time (min)
0
10
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30
40
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Rel
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NL:8.53E6
TIC MS 0128bms2
c:\documents and settings\...\0128bms2 1/28/2004 3:09:24 PM BMS (10.58mg/mL)+ DPPC (0.500035mg/mL)2 TEES: UV/ELSD, LCQ-APCI*TSQ-ESI, 20mM NH4OCOH@20uL/min Sheath
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL: 1.27E7
m/z= 634.1-635.1 MS BMS +ve APCI Dual Quan_b
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL:3.84E5m/z= 817.2-818.2 MS 0128bms2
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL: 6.18E7
m/z= 632.1-633.1 MS BMS +ve APCI Dual Quan_b
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL:1.44E6m/z= 815.2-816.2 MS 0128bms2
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL: 5.71E7
m/z= 606.1-607.1 MS BMS +ve APCI Dual Quan_b
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL:8.90E5m/z= 789.2-790.2 MS 0128bms2
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL: 9.40E7
m/z= 604.1-605.1 MS BMS +ve APCI Dual Quan_b
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL:2.00E6m/z= 787.2-788.2 MS 0128bms2
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL: 1.69E7
m/z= 522.0-523.0 MS BMS +ve APCI Dual Quan_b
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL:4.18E5m/z= 705.1-706.1 MS 0128bms2
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL: 8.62E7
m/z= 520.0-521.0 MS BMS +ve APCI Dual Quan_b
26 28 30 32 34 36 38 40 42 44Time (min)
0
50
100 NL:3.17E6m/z= 703.1-704.1 MS 0128bms2
APCI-MSAPCI-MS ESI-MSESI-MS24:0 DSM 24:0 DSMm/z 634.6 m/z 817.7
24:1 DSM 24:1 DSMm/z 632.6 m/z 815.7
22:0 DSM 22:0 DSMm/z 606.6 m/z 789.7
22:0 SM 22:0 SMm/z 604.6 m/z 787.7
16:0 DSM 16:0 DSMm/z 522.5 m/z 705.6
16:0 SM 16:0 SMm/z 520.5 m/z 703.6
24:0 SM 24:0 SM
Quantification of Sphingolipids
By APCI-MS and ESI-MS
(if time allows)
Quantification of Sphingolipids
By APCI-MS and ESI-MS
(if time allows)
DHS SPMFA 16:0 17:0 18:0 19:0 20:0 16:1 17:1 18:1 19:1 20:1
14:0 0.04 0.0415:0 0.0216:0 0.65 0.00 1.4417:0 0.1218:0 19.60 0.88 0.19 0.10 40.35 0.89 6.8318:1 0.86 0.7319:0 0.84 1.0720:0 7.31 0.09 0.08 3.2420:1 0.94 0.76 0.0421:0 0.64 0.02 0.7622:0 17.11 6.40 0.0222:1 2.92 2.0723:0 3.24 0.03 2.11 0.0223:1 1.09 0.56 0.03 0.69 0.0424:0 0.08 15.28 0.06 7.49 0.02 0.0424:1 0.13 23.30 0.35 19.78 0.11 0.0625:0 1.23 0.02 0.92 0.0025:1 0.88 1.81 0.0126:0 0.51 0.00 0.2826:1 1.48 1.28
Total DHS = 100% Total SPM = 100%
Bovine Brain Sphingolipid Molecular Species by APCI-MS
Bovine Brain Sphingolipid Molecular Species by APCI-MS
FA Mole % DHS-LCB %14:0 0.1 16:0 0.215:0 0.0 17:0 1.116:0 1.3 18:0 97.417:0 0.1 19:0 0.418:0 43.4 20:0 0.918:1 0.819:0 1.0 SPM-LCB %20:0 4.1 16:1 0.320:1 0.8 17:1 0.221:0 0.8 18:1 91.422:0 8.3 19:1 1.122:1 2.2 20:1 7.123:0 2.323:1 0.9 Sphingolipid %24:0 8.9 DHS 17.724:1 20.6 SPM 82.325:0 1.0
25:1 1.726:0 0.3 Sphingolipid #26:1 1.3 DHS 25
Sum = 100.0 SPM 38
DHS SPMFA 16:0 17:0 18:0 19:0 20:0 16:1 17:1 18:1 19:1 20:1
14:0 0.11 0.0715:0 0.0416:0 1.16 0.01 2.7117:0 0.1718:0 24.55 1.31 0.36 0.15 48.31 0.22 8.2518:1 0.36 0.3919:0 0.59 0.2620:0 10.90 0.11 0.02 3.9220:1 0.56 0.15 0.0421:0 0.45 0.01 0.3122:0 16.79 6.02 0.0122:1 3.29 1.8223:0 2.87 0.02 1.47 0.0123:1 1.23 1.07 0.01 0.84 0.0124:0 0.08 14.28 0.03 6.35 0.01 0.0424:1 0.08 17.77 0.15 15.51 0.04 0.0425:0 0.58 0.02 0.47 0.0025:1 0.38 0.59 0.0126:0 0.54 0.00 0.3026:1 0.94 0.84
Total DHS = 100% Total SPM = 100%
Bovine Brain Sphingolipid Molecular Species by ESI-MS
Bovine Brain Sphingolipid Molecular Species by ESI-MS
FA Mole % DHS-LCB %14:0 0.1 16:0 0.215:0 0.0 17:0 1.216:0 2.4 18:0 97.117:0 0.1 19:0 0.218:0 51.8 20:0 1.318:1 0.419:0 0.3 SPM-LCB %20:0 5.3 16:1 0.520:1 0.3 17:1 0.221:0 0.3 18:1 90.622:0 7.9 19:1 0.322:1 2.1 20:1 8.523:0 1.723:1 1.1 Sphingolipid %24:0 7.8 DHS 17.524:1 16.0 SPM 82.525:0 0.5
25:1 0.626:0 0.3 Sphingolipid #26:1 0.9 DHS 25
Sum = 100.0 SPM 39
ConclusionConclusion
• Dihydrosphingomyelin is a dietary component!
• APCI-MS can provide fragments to
identify the backbone and fatty amide.
• ESI-MS provides the molecular weight.
• Dual Parallel Mass Spectrometers
reduce or eliminate ambiguity.
• 31P-NMR proves DSM is present.
• Dihydrosphingomyelin is a dietary component!
• APCI-MS can provide fragments to
identify the backbone and fatty amide.
• ESI-MS provides the molecular weight.
• Dual Parallel Mass Spectrometers
reduce or eliminate ambiguity.
• 31P-NMR proves DSM is present.
Thank You for your Attention!Thank You for your Attention!
Wm. Craig Byrdwell, PhD
USDA, ARS, BHNRC
Food Composition Lab
Byrdwell@ba.ars.usda.gov
http://www.Byrdwell.com
Wm. Craig Byrdwell, PhD
USDA, ARS, BHNRC
Food Composition Lab
Byrdwell@ba.ars.usda.gov
http://www.Byrdwell.com
www.Sphingomyelin.comwww.PhosphatidylCholines.com
www.PhosphatidylEthanolamines.comwww.PhosphatidylSerines.com
www.PhosphatidylGlycerols.comwww.PhosphatidylInositols.com
www.Triacylglycerols.com
www.Sphingomyelin.comwww.PhosphatidylCholines.com
www.PhosphatidylEthanolamines.comwww.PhosphatidylSerines.com
www.PhosphatidylGlycerols.comwww.PhosphatidylInositols.com
www.Triacylglycerols.com
You may find these Byrdwell.com Websites helpful:
You may find these Byrdwell.com Websites helpful:
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