Transcript MALDI-ToF Mass Spectrometry of Phosphorylated Lipids in Tear Samples Richard B. Cole

MALDI-ToF Mass Spectrometry of
Phosphorylated Lipids in Tear Samples
Richard B. Cole1,*, Bryan M. Ham1, Jean T. Jacob2
1. Dept. of Chemistry, University of New Orleans, New Orleans, LA
2. Dept. of Ophthalmology, LSU-HSC, New Orleans, LA
Objectives
• Develop new MALDI-ToF matrix for
improved MS detection of phospholipids
• Develop novel method for efficient
extraction of low-level phospholipids
• Apply above methodology to identify and
compare phosphorylated lipids in normal
and “dry eye” model tears
Synthesis of solid ionic crystal
matrix for MALDI
714
551
[16:0/16:0-PE+Na]+
[PE+H]+
537
692
564
736
693
580
% Intensity
% Intensity
100
90 (a)
80
70
60
50
40
30
20
10
0
500
737
660
740
820
900
100
90 (d)
80
70
60
50
40
30
20
10
0
500
605
603
551
564
621
580
660
[16:0/16:0-PE+Na]+
% Intensity
% Intensity
714
552
[PE+H]+
564
736
692
580
660
740
740
820
900
100
90 (e)
80
70
60
50
40
30
20
10
0
500
[16:0/16:0-PE+H]+
693
564
714 730
580
660
% Intensity
% Intensity
(c)
622
643
659
580
660
740
Mass (m/z)
740
820
900
Mass (m/z)
621
551
900
692
Mass (m/z)
100
90
80
70
60
50
40
30
20
10
0
500
820
Mass (m/z)
551
(b)
693
639
Mass (m/z)
100
90
80
70
60
50
40
30
20
10
0
500
[16:0/16:0-PE+H]+
692
820
900
100
90
80 (f)
70
60
50
40
30
20
10
0
500
692
551
[16:0/16:0-PE+H]+
564
693
552
694
621
580
660
740
820
900
Mass (m/z)
Comparison of six MALDI matrixes for the analysis of 16:0/16:0-phosphatidylethanolamine (PE) in positive ion mode: (a) DHB; (b) -CHCA/DHB plus TFA; (c) PNA;
(d) PNA plus TFA; (e) PNA-butyric acid solid ionic crystal; (f) PNA-butyric acid plus TFA.
Zwitterionic Phosphorylated Lipids
O
O
O
N
Anionic Phosphorylated Lipids
P
O
O
O
O
R1
H
Phosphatidylcholine (PC)
H
O
O
O
O
O
O
O
Na
P
O
P
H
O
O
O
N
H
H
O
O
O
N
O
H
O
O
R2
Phosphatidylserine (PS)
R1
R2
R2
O
O
O
HO
R1
P
O
Phosphatidylethanolamine (PE)
N
O
O
Na
O
O
O
O
O
Phosphatidic acid (PA)
R1
R2
O
P
O
O
O
R1
OH
O
HO
O
O
O
P
O
Platelet activating factor (PAF)
N
O
O
Na
O
O
O
O
O
Phosphatidylglycerol (PG)
R1
R2
O
P
O
O
O
R1
H
OH
HO
L yso phosphatidylcholine (L yso PC)
O
N
R1
O
H
H
H
P
OH
O
H
Na
Phosphatidylinositol (PI )
N
R2
H
O
Sphingomyelin (SM )
O
O
O
H
P
O
HO
HO
OH
O
OH
O
O
O
R2
O
R1
% Intensity
% Intensity
100 (a)
90
80 (a)
70
60
50
40
30
59
20
10
0
50
100 (b)
90
(b)
123
80
70
60 122
50
139
40
30
124
20
10
0
110
109
123
122
1.7E+4
108
124
+
[PNA+H]
[PNA+H]
+
139
229
110
100
150
200
496
Mass (m/z)
[16:0-LysoPC+H]+
[16:0LysoPC+H]+
250
[14:0/14:0-DMPC+H]+
0
300
3.9E+4
678
[14:0/14:0DMPC+H]+
679
229
497
680
248
386
524
662
0
800
Mass (m/z)
MALDI-TOF mass spectra of: (a) para-nitroaniline/butyric acid matrix preparation
showing background peaks originating from matrix; (b) 2-component mixture of
16:0-lyso PC and 14:0/14:0-DMPC showing protonated 16:0-lyso PC at m/z 496,
and protonated 14:0/14:0-DMPC at m/z 678 using PNA-butyric acid matrix.
PNA/butyric acid solid ionic crystal matrix
• High sensitivity, simultaneous detection of
phosphorylated lipids in mixtures
• Reliable appearance of MH+ of lipids containing PC
head group
• Reliable appearance of [M+Na]+ adducts of anionic
phospholipids
• Potential for use as matrix in MALDI imaging
Extraction of phosphorylated lipids
• Developed method based on use of immobilized metal
ion affinity chromatography (IMAC) media (“ZipTip”,
Millipore Inc., Bedford, MA) originally intended for
phosphopeptides
• Binding solution changed from 0.1% acetic acid (aq) to
0.1% acetic acid in 1:1 MeOH:ACN
• Elution solution changed from 0.3 N NH4OH (aq) to 0.3
N NH4OH in 1:1 MeOH:ACN
• IMAC media soluble in CHCl3, so CHCl3 must be
removed prior to contact with ZipTip
% Intensity
100
90
80
70
60
50
40
30
20
10
0
450
678
496
1.7E+4
733
731
692
510
570
630
690
0
750
Mass (m/z)
Efficient extraction, isolation, clean-up and recovery of an
equimolar 4-component phosphorylated lipid standard
mixture using IMAC ZipTip.
Protonated 16:0-lyso phosphatidylcholine at m/z 496,
protonated dimyristyl phosphatidylcholine at m/z 678,
protonated dipalmitoyl phosphatidylethanolamine at m/z 692, and
protonated 16:0-sphingomyelin at m/z 731.
Outer Tear Film Lipid Layer
McCulley, J.P., Shine, W. Tr. Am. Ophth. Soc. 1997, 95, 79-93
“Dry Eye” Model
• Dry eye syndrome afflicts 12 million in US
• Tear samples obtained from normal & dry
eyes of female New Zealand white rabbits
• Experimental dry eye induced by removal of
main and accessory lacrimal glands and
nictitating membranes
• All animal studies conducted in accordance
with Association for Research in Vision
and Ophthalmology guidelines
% Intensity
100
90
80 455
70
60
50
40
30
20
10
0
450
610
638
(a) Normal eye tear extracted lipids
522
537
550
494
540
630
720
810
900
Mass (m/z)
% Intensity
(b) Dry eye tear extracted lipids
100
90
80
70
60
50
40
30
20
10
0
550
731
(c) Sphingomyelin standard
813
605
753 759
621
769 787
637 659
643
610
670
730
Mass (m/z)
790
850
% Intensity
100
90
80
70
60
50
40
30
20
10
0
605
[M+H]+
N
O
O
P
HO
N
O
O
O
O
O
P
P
HO
HO
OH
OH
335
184
151
[M+H-O]+
280
272
0
[M+H-C17H34O2]+
400
303
454
Mass (m/z)
PSD of m/z 605 from sphingomyelin standard.
589
550
606
758
O
O
N
P
OH
O
O
O
O
O
(CH2)11CH3
P
O
O
H
N
mol. wt. = 604
O
O
O
N
P
OH
O
O
O
O
OH
O
(CH2)11CH3
P
O
O
H
N
mol. wt. = 620
O
O
O
N
P
O
OH
O
O
O
P
O
mol. wt. = 636
OH
O
O
O
(CH2)11CH3
H
N
O
Proposed structures of pyrophosphate sphingomyelins
(a) PSD of m/z 678 (normal eye, also in dry)
N
O
HO
P
O
OH
(b) PSD of m/z 828 (dry eye only)
H3 N
MI-NL of 87 Da
m/z 741
CH2
O
C
O
Table 1. Major phosphorylated analyte peaks observed in the MALDI-TOF mass spectra
for both normal and dry eye rabbit tears with tentative assignments.
Molecular Ion
MI
Normal
Eye**
Dry
Eye**
[M+H]+, m/z 494
major
major
M/z 522 – unidentified
[M+H]+
major
major
M/z 550 – unidentified
[M+H]+
major
major
Pyrophosphate SM C22H46N2O11P2
[M+H]+, m/z 577
major
major
Pyrophosphate SM C24H50N2O11P2
[M+H]+, m/z 605
-
major
Pyrophosphate SM C24H50N2O12P2
[M+H]+, m/z 621
-
major
Pyrophosphate SM C24H50N2O13P2
[M+H]+, m/z 637
minor
major
Pyrophosphate SM C24H50N2O13P2Na
[M+Na]+, m/z 659
minor
major
M/z 610 – unidentified
[M+H]+
major
major
M/z 638 – unidentified
[M+H]+
major
-
M/z 642 – unidentified
[M+H]+
minor
major
[M+H]+, m/z 678
minor
minor
[M+H]+
minor
minor
[M+2Na-H]+, m/z 828
-
minor
M/z 866 – unidentified
[M+H]+
minor
-
M/z 886 – unidentified
[M+H]+
-
minor
M/z 936 – unidentified
[M+H]+
minor
Lipid in Tear Extract
C14:1-2:0 PAF or C16:1 Lyso-PC
C14:0-14:0 PC
M/z 695 – unidentified
16:0-20:4 PS C42H73NO10PNa
-
**The assignment of major or minor to the presence of the phospholipids is qualitative and is derived from the relative intensity of the peak in mass spectra.
- = not detected
Conclusions
• Two major SM peaks (m/z 605, 621) detected in dry eye
tears were not found in normal tears.
• Two other SM peaks (m/z 637, 659) found as major
peaks in dry eye were minor in normal eye.
• A minor PS peak (m/z 828) appeared in dry eye but was
absent in normal eye.
• Increased presence of phospholipids SM and PS in dry
eye could indicate over-stimulation of meibomian gland
and release of excess phospholipids to stabilize tear film.
Financial Support
• Louisiana Board of Regents Health Excellence Fund
HEF(2001-06)-08. (RBC)
• USPHS grants R03EY014021 (JTJ)
• P30EY002377 (LSU Eye Center Core grant)
• National Eye Institute, National Institutes of Health,
Bethesda, Maryland
• Research to Prevent Blindness, Inc., New York, New
York (LSU Eye Center).