1 / 19

MALDI-ToF Mass Spectrometry of Phosphorylated Lipids in Tear Samples

MALDI-ToF Mass Spectrometry of Phosphorylated Lipids in Tear Samples Richard B. Cole 1,* , Bryan M. Ham 1 , Jean T. Jacob 2 Dept. of Chemistry, University of New Orleans, New Orleans, LA 2. Dept. of Ophthalmology, LSU-HSC, New Orleans, LA. Objectives.

saxton
Download Presentation

MALDI-ToF Mass Spectrometry of Phosphorylated Lipids in Tear Samples

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. 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

  3. Synthesis of solid ionic crystal matrix for MALDI

  4. (d) [16:0/16:0-PE+H]+ (a) [16:0/16:0-PE+Na]+ [PE+H]+ (b) [16:0/16:0-PE+Na]+ (e) [16:0/16:0-PE+H]+ [PE+H]+ (c) [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.

  5. O O O N P O O R 1 O O O O R 2 O H O P h o s p h a t i d y l c h o l i n e ( P C ) O N P O H O O R 1 H O O R O 2 H O N a O N P P h o s p h a t i d y l s e r i n e ( P S ) O H O O R 1 H O O O R O 2 H O P O O R O 1 O P h o s p h a t i d y l e t h a n o l a m i n e ( P E ) N a O R 2 P h o s p h a t i d i c a c i d ( P A ) O O O O N P O O R O H 1 O O O O H O O P O O R 1 O O P l a t e l e t a c t i v a t i ng f a c t o r ( P A F ) O R 2 N a O O P h o s p h a t i d y l g l y c e r o l ( P G ) O O N P O O R H O H 1 O O H H O H H O L y s o p h o s p h a t i d y l c h o l i n e ( L y s o P C ) O H O O H O P H O H O O R 1 O H O O H H O O R 2 N a R N P 1 O P h o s p h a t i d y l i n o s i t o l ( P I ) O O N R 2 H O S p h i n g o m y e l i n ( S M ) Zwitterionic Phosphorylated Lipids Anionic Phosphorylated Lipids

  6. (a) (a) [PNA+H]+ [PNA+H]+ (b) [14:0/14:0-DMPC+H]+ [16:0-LysoPC+H]+ (b) [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.

  7. 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

  8. 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

  9. 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.

  10. Outer Tear Film Lipid Layer McCulley, J.P., Shine, W. Tr. Am. Ophth. Soc. 1997, 95, 79-93

  11. “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

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

  13. [M+H]+ [M+H-C17H34O2]+ [M+H-O]+ PSD of m/z 605 from sphingomyelin standard.

  14. Proposed structures of pyrophosphate sphingomyelins

  15. N H N 3 O C H O 2 P O C H O O H O (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

  16. 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

  17. 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.

  18. 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).

More Related