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Ben J. Glasgow, Austin W. Dean; Mass Spectrometric Identification of Phospholipids Extracted from Human Tears and Tear Lipocalin. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2701.
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Previously, we identified phospholipids by thin layer chromatography in tears and as native ligands of purified tear lipocalin (Curr. Eye Res. 14; 1995:363-372). Despite the advent of advanced mass spectrometric techniques with enhanced ability to define the precise phospholipid molecules, the presence and amount of phospholipids in tears are still disputed. Our purpose was to identify phosphocholine lipids in stimulated human tears and determine the species bound to tear lipocalin or other proteins by mass spectrometry.
Stimulated human tears were collected and pooled. Tear proteins were separated by liquid chromatography using a Superdex 75 hi load 16/60 preparative grade column (GE Healthcare). The column was prewashed in 30% acetonitrile and equilibrated in 0.050 M Tris-HCl pH 8.4, 0.1 M NaCl. Tears were separated under isocratic conditions at a flow rate of 1 mL/min. Chromatographic separation was confirmed by SDS tricine gradient polyacrylamide gel electrophoresis. Protein fractions were extracted with chloroform/methanol, dried under nitrogen and analyzed with electrospray ionization, MS/MS triple quadrupole mass spectrometry (Agilent 6460) in precursor ion scan mode for select leaving groups. For comparison and quantitation, integrated ion counts were derived from standard curves of authentic compounds of phosphatidylcholine and phosphatidylserine.
Linear approximation was possible from integration of the mass spectrometrically obtained ion peaks at 760 Da for the phosphatidylcholine standard. The amount from the major intact species, 758.6 Da PC(34:2), in tears was estimated to be 194 ng/ml. Ten other monoisotopic phosphocholine bearing lipids were found in stimulated tears. A peak at 703.3 Da was assigned to a sphingomyelin species. Four peaks with high signal intensity were discovered in the 490-540 m/z range. All 4 were assigned to lysophosphatidylcholine species and accounted for about 80% of the signal intensity and total integrated ion count. The M-H+ species at m/z= 496.3 accounted for almost 60% of the signal intensity. Only the tear lipocalin bearing gel filtration fraction showed extractable lipid. Linear regression (R2 =0.9919) of the integrated ion count revealed that the extracted tear lipocalin fraction contained about 104 ng/ml. While the intact phospholipids bound to tear lipocalin corresponded precisely in mass and relative signal intensity to that found in tears,we did not identify species in the 490-540 Da range in any of the gel filtration fractions.
Phospholipids, especially lysophospholipids, are present in tears. The higher mass intact phosphatidylcholines in tears are native ligands of tear lipocalin.
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