Mass spectrometric identification of phospholipids in human tears and tear lipocalin.

ABSTRACT

PURPOSE: The purpose of this article was to identify by mass spectrometry phosphocholine lipids in stimulated human tears and determine the molecules bound to tear lipocalin or other proteins. METHODS: Tear proteins were separated isocratically from pooled stimulated human tears by gel filtration fast performance liquid chromatography. Separation of tear lipocalin was confirmed by SDS tricine gradient PAGE. Protein fractions were extracted with chloroform/methanol and analyzed with electrospray ionization MS/MS triple quadrupole mass spectrometry in precursor ion scan mode for select leaving groups. For quantification, integrated ion counts were derived from standard curves of authentic compounds of phosphatidylcholine (PC) and phosphatidylserine. RESULTS: Linear approximation was possible from integration of the mass spectrometrically obtained ion peaks at 760 Da for the PC standard. Tears contained 194 ng/mL of the major intact PC (342), m/z 758.6. Ten other monoisotopic phosphocholines were found in tears. A peak at 703.3 Da was assigned as a sphingomyelin. Four lysophosphatidylcholines (m/z 490-540) accounted for about 80% of the total integrated ion count. The [M+H](+) compound, m/z 496.3, accounted for 60% of the signal intensity. Only the tear lipocalin-bearing fractions showed phosphocholines (104 ng/mL). Although the intact phospholipids bound to tear lipocalin corresponded precisely in mass and relative signal intensity to that found in tears, we did not identify phosphocholines between m/z 490 and 540 in any of the gel-filtration fractions. CONCLUSIONS: Phospholipids, predominantly lysophospholipids, are present in tears. The higher mass intact PCs in tears are native ligands of tear lipocalin.