MALDI-ToF Mass Spectrometry of Phosphorylated Lipids in Tear Samples Richard B. Cole 1,*, Bryan M. Ham 1, Jean T. Jacob 2 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

[PE+H] + (a) [16:0/16:0-PE+Na] + (b) [PE+H] + [16:0/16:0-PE+Na] + (c) [16:0/16:0-PE+H] + (d) [16:0/16:0-PE+H] + (e) [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 LipidsAnionic Phosphorylated Lipids O P O O O OR 1 O OR 2 O N O P O O O OR 1 O OR 2 O N H H H O P O O O OR 1 O O O N O P O O O OR 1 O OH N O P O O O N R 2 O N H OH R 1 Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Platelet activating factor (PAF) Lyso phosphatidylcholine (Lyso PC) Sphingomyelin (SM) O P O O O OR 1 O OR 2 O N H H H Na O O Phosphatidylserine (PS) HO P O O O OR 1 O OR 2 O Na Phosphatidic acid (PA) O P O O O OR 1 O OR 2 O HO OH Na Phosphatidylglycerol (PG) O P O O O OR 1 O OR 2 O H HO H HO H H OH H HO OH H Phosphatidylinositol (PI) Na

(a) (b) [16:0- LysoPC+H] + [14:0/14:0- DMPC+H] + [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 NH 4 OH (aq) to 0.3 N NH 4 OH in 1:1 MeOH:ACN IMAC media soluble in CHCl 3, so CHCl 3 must be removed prior to contact with ZipTip

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, 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

(c) Sphingomyelin standard (a) Normal eye tear extracted lipids (b) Dry eye tear extracted lipids

[M+H-C 17 H 34 O 2 ] + [M+H] + [M+H-O] + PSD of m/z 605 from sphingomyelin standard.

Proposed structures of pyrophosphate sphingomyelins

MI-NL of 87 Da m/z 741 (a) PSD of m/z 678 (normal eye, also in dry) (b) PSD of m/z 828 (dry eye only) N O P O OH HO OC O H 3 N CH 2

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 ExtractMolecular Ion MI Normal Eye** Dry Eye** C14:1-2:0 PAF or C16:1 Lyso-PC[M+H] +, m/z 494major M/z 522 – unidentified[M+H] + major M/z 550 – unidentified[M+H] + major Pyrophosphate SM C 22 H 46 N 2 O 11 P 2 [M+H] +, m/z 577major Pyrophosphate SM C 24 H 50 N 2 O 11 P 2 [M+H] +, m/z 605 -major Pyrophosphate SM C 24 H 50 N 2 O 12 P 2 [M+H] +, m/z 621 -major Pyrophosphate SM C 24 H 50 N 2 O 13 P 2 [M+H] +, m/z 637minormajor Pyrophosphate SM C 24 H 50 N 2 O 13 P 2 Na[M+Na] +, m/z 659minormajor M/z 610 – unidentified[M+H] + major M/z 638 – unidentified[M+H] + major - M/z 642 – unidentified[M+H] + minormajor C14:0-14:0 PC[M+H] +, m/z 678minor M/z 695 – unidentified[M+H] + minor 16:0-20:4 PS C 42 H 73 NO 10 PNa[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( )-08. (RBC) USPHS grants R03EY (JTJ) P30EY (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).