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, LA2. 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

500 580 660 740 820 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

ns

ity

714 551

537

692 564 736

693 737

[PE+H]+

(a)

500 580 660 740 820 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% In

ten

sity

551

714

552

564 736

692

[16:0/16:0-PE+Na]+

(b)

[PE+H]+

[16:0/16:0-PE+Na]+

500 580 660 740 820 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% In

tens

ity

621

622 551 643

659

(c)

500 580 660 740 820 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

ns

ity

605

692

603 551 564 693 639

621

[16:0/16:0-PE+H]+ (d)

500 580 660 740 820 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

ns

ity

692

693

564 730 714

[16:0/16:0-PE+H]+(e)

500 580 660 740 820 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

ns

ity

692 551

564 693

552

694 621

[16:0/16:0-PE+H]+(f)

Comparison of six MALDI matrixes for the analysis of 16:0/16:0-phosphatidyl-ethanolamine (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 Anionic Phosphorylated Lipids

OP

O

OO

O R 1

O

O R 2

O

N

OP

O

OO

O R 1

O

O R 2

O

N

H

HH

OP

O

OO

O R 1

O

O

O

N

OP

O

OO

O R 1

O

OH

N

OP

O

OO

N R 2

O

N

H

O H

R 1

Phosphatidylcholine (PC)

Phosphatidylethanolamine (PE)

Platelet activating factor (PAF)

Lyso phosphatidylcholine (Lyso PC)

Sphingomyelin (SM )

OP

O

OO

O R 1

O

O R 2

O

N

H

HH

N a

OO

Phosphatidylserine (PS)

H OP

O

OO

O R 1

O

O R 2

O

N a

Phosphatidic acid (PA)

OP

O

OO

O R 1

O

O R 2

O

H O

O H

N a

Phosphatidylglycerol (PG)

OP

O

OO

O R 1

O

O R 2

O

H

H O

H

HO

H

H

O HH

H O

O H

H

Phosphatidylinositol (PI )Na

(a)

(b) [16:0-LysoPC+H]+ [14:0/14:0-

DMPC+H]+

[PNA+H]+

50 100 150 200 250 300 Mass (m/z)

0

1.7E+4

0 10 20 30 40 50 60 70 80 90

100

% I

nte

nsi

ty

109

124 229

110

59

139

123

108

122

110 248 386 524 662 800 Mass (m/z)

0

3.9E+4

0 10 20 30 40 50 60 70 80 90

100

% I

nte

nsi

ty

496

678 123

122 679

229 497 124

680

139

[PNA+H]+

[16:0-LysoPC+H]+ [14:0/14:0-DMPC+H]+

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.

(a)

(b)

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

450 510 570 630 690 750 Mass (m/z)

0

1.7E+4

0 10 20 30 40 50 60 70 80 90

100

% I

nte

nsi

ty

678 496

692

731 733

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.

McCulley, J.P., Shine, W. Tr. Am. Ophth. Soc. 1997, 95, 79-93

Outer Tear Film Lipid Layer

“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

550 610 670 730 790 850 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

nsi

ty

731

813 605

759 621

787 769 637 643 659

753

450 540 630 720 810 900 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

nsi

ty

610 638

455 522

550 494

537

(c) Sphingomyelin standard

(a) Normal eye tear extracted lipids

(b) Dry eye tear extracted lipids

0 151 303 454 606 758 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% I

nte

nsi

ty

605

335 280 184

272 550 400 589

N

O

PO

OHO

O

PO

OHHO

N

O

P

O

OHHO

[M+H-C17H34O2]+

[M+H]+

[M+H-O]+

PSD of m/z 605 from sphingomyelin standard.

OP

O

OO

N

O

NOH

(CH2)11CH3OP

O

OO H

O

OP

O

OO

N

O

NOH

(CH2)11CH3O

P

O

OO H

OOH

OP

O

OO

N

O

NOH

(CH2)11CH3O

P

O

OO H

OOH

O

mol. wt. = 604

mol. wt. = 620

mol. wt. = 636

Proposed structures of pyrophosphate sphingomyelins

MI-NL of 87 Dam/z 741

(a) PSD of m/z 678 (normal eye, also in dry)

(b) PSD of m/z 828 (dry eye only)

N

O

PO

OHHO

O C

O

H3N

CH2

Table 1. Major phosphorylated analyte peaks observed in the MALDI-TOF mass spectra for both normal and dry eye rabbit tears with tentative assignments.

**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

Lipid in Tear Extract Molecular IonMI

NormalEye**

DryEye**

C14:1-2:0 PAF or C16:1 Lyso-PC [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

C14:0-14:0 PC [M+H]+, m/z 678 minor minor

M/z 695 – unidentified [M+H]+ minor minor

16:0-20:4 PS C42H73NO10PNa [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 -

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).