MALDIToF Mass Spectrometry of

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• MALDI-ToF Mass Spectrometry of
• Phosphorylated Lipids in Tear Samples
• Richard B. Cole1,, Bryan M. Ham1, Jean T. Jacob2
• Dept. of Chemistry, University of New Orleans,
  New Orleans, LA
• 2. Dept. of Ophthalmology, LSU-HSC, New Orleans,
  LA

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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

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Synthesis of solid ionic crystal matrix for MALDI
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160/160-PEH
(d)
(a)
160/160-PENa
PEH
(b)
160/160-PENa
160/160-PEH
(e)
PEH
(c)
160/160-PEH
(f)
Comparison of six MALDI matrixes for the analysis
of 160/160-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.
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Zwitterionic Phosphorylated Lipids
Anionic Phosphorylated Lipids
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(a)
(a)
PNAH
PNAH
(b)
140/140-DMPCH
160-LysoPCH
(b)
160-LysoPCH
140/140-DMPCH
MALDI-TOF mass spectra of (a) para-nitroaniline/b
utyric acid matrix preparation showing background
peaks originating from matrix (b) 2-component
mixture of 160-lyso PC and 140/140-DMPC
showing protonated 160-lyso PC at m/z 496, and
protonated 140/140-DMPC at m/z 678 using
PNA-butyric acid matrix.
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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 MNa adducts of anionic
  phospholipids
• Potential for use as matrix in MALDI imaging

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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 11 MeOHACN
• Elution solution changed from 0.3 N NH4OH (aq) to
  0.3 N NH4OH in 11 MeOHACN
• IMAC media soluble in CHCl3, so CHCl3 must be
  removed prior to contact with ZipTip

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Efficient extraction, isolation, clean-up and
recovery of an equimolar 4-component
phosphorylated lipid standard mixture using IMAC
ZipTip. Protonated 160-lyso phosphatidylcholine
at m/z 496, protonated dimyristyl
phosphatidylcholine at m/z 678, protonated
dipalmitoyl phosphatidylethanolamine at m/z 692,
and protonated 160-sphingomyelin at m/z 731.
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Outer Tear Film Lipid Layer
McCulley, J.P., Shine, W. Tr. Am. Ophth. Soc.
1997, 95, 79-93
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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

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(a) Normal eye tear extracted lipids
(b) Dry eye tear extracted lipids
(c) Sphingomyelin standard
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MH
MH-C17H34O2
MH-O
PSD of m/z 605 from sphingomyelin standard.
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Proposed structures of pyrophosphate
sphingomyelins
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(a) PSD of m/z 678 (normal eye, also in dry)
(b) PSD of m/z 828 (dry eye only)
MI-NL of 87 Da m/z 741
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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
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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.

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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).

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