1,4). The lipids in the outermost lipid layer originate from meibum, secretions from the meibomian glands situated posterior to the eyelashes on the eyelid margin (1). Changes in the composition and/or quantity of the lipid secretions from the meibomian glands lead to evaporative dry eye disease (DED) (5, 6), a disorder characterized by symptoms of dryness and visual disturbance that alter quality of life (7). DED affects nearly 5 million people aged 50 years and older in the United States and likely tens of millions more with less severe symptoms. The prevalence ranges from 5% to 50% worldwide depending on age and definition (7).A clear understanding of the lipid composition of meibum would help to elucidate the mechanisms underlying DED, thereby facilitating the development of effective treatments. Meibum secretions have been shown to be almost exclusively composed of lipids (8). These lipids are predominantly neutral lipids and include the wax esters (WEs), cholesteryl esters (CEs), diesters, and triacylglycerols (TGs), as well as smaller amounts of polar lipids, such as FFAs and (O-acyl)--hydroxy fatty acids (OAHFAs) (9-14). However, our knowledge of the exact lipid composition of meibum, including the specific isomeric species present and the identities of additional lipids present in low abundance, remains lacking (15).Various mass spectrometric methods (9-14) have been applied to characterize the lipid composition of meibum. In 1981, Nicolaides et al. (10) were the first to report a comprehensive study of meibum . They reported the different classes of lipids present, along with the percentages of Abstract The lipid composition of human meibomian gland secretions (meibum) has been analyzed using both targeted and untargeted mass spectrometric approaches, each of which has its advantages and disadvantages. Herein we report the results of shotgun lipidomic profiling of human meibum using a new approach that combines the advantages of targeted and untargeted analyses to yield highly sensitive and comprehensive profiles. Samples containing an estimated 7-13 µg (8-16 nL) of human meibum lipids were analyzed using MS/MS all , an untargeted approach for MS/MS. Using MS/MS all with ESI and successive polarity switching, we obtained tandem mass spectra in both modes at every 1 Da step for all ions in the m/z 200-1,200 range. In approximately 12 min, a total of 2 MS spectra and 2,000 MS/MS spectra were acquired for each sample, from which targeted analysis information was extracted. This approach allowed for the comprehensive and highly sensitive detection of meibum lipids, including species low in abundance. Altogether, more than 600 unique lipid molecular species were identified in meibum, 3 times more than previously reported in untargeted analyses of meibum samples. This untargeted MS and MS/MS all approach may be extended to other biological systems for the detection of lipids with sensitivity comparable to targeted analysis.-Chen, J., and K. K. Nichols. Comprehensive shotgun lipidomics of human meibomian gl...