Novel pathomechanisms of impairment of native tear protein complexes in aqueous deficient dry eye syndrome

Abstract

Aims/Purpose: Dry eye syndrome (DES) is a common yet deplorable disease of the ocular surface with high global prevalence. The pathomechanisms underlying the impairment of the native tear protein complexes remain unexplored. This study established a quantitative proteomics approach to characterize the novel protein complexes and unravel their functional roles in tears of DES patients.Methods: Tear samples were collected using Schirmer's strips from aqueous‐deficient DES (DRY, n = 16) and healthy (CTRL, n = 16) subjects following comprehensive DES clinical examinations. The native tear protein complex profiles were elucidated employing a customized blue native gel electrophoretic (BN‐PAGE) combined with mass spectrometry (MS)‐based proteomics strategy. In‐silico bioinformatics tools were employed for gene annotation analysis of the complexes.Results: Five major native protein complexes designated as complex I (cI, 800 kDa), II (cII, 400 kDa), III (cIII, 240 kDa), IV (cIV, 140 kDa) and V (cV, 66 kDa) were characterized for the first time in human tears. Interestingly, a cluster of immune‐related proteins identified in cI (e.g., IGHA1) were annotated to complement pathways and was found to be significantly increased in abundance in DRY vs. CTRL (p = 8.2 × 10−3). Likewise, increased abundance of another cluster of proteins annotated to inflammatory response (e.g. S100A9) in DRY (p = 1.7 × 10−2) was associated to cIII. On the contrary, cII was largely composed of lacrimal‐specific proteins (e.g., LYZ), which was found to be decreased in abundance in DRY (p = 2.4 × 10−2). Similarly, decrement of antimicrobial proteins in cIV (e.g., LACRT) was observed in DRY (p = 1.7 × 10−2). The expression of a cluster of carrier proteins involved in protein–protein interactions (e.g., ALB) was annotated in cV, which was increased in DRY (p = 4.4 × 10−3). Noteworthy, 35 new DES markers were identified with this methodological approach.Conclusions: Collectively, a tear‐customized BN‐PAGE‐MS analysis was instrumental to decipher the multifaceted biological functions of novel native protein complexes in human tears. For the first time, the intricate molecular mechanistic changes underlying these protein complexes in DES were unravelled, which could be instrumental for potential development of therapeutic strategies in ameliorating DES.