Investigation of tear protein composition in healthy and dry eye subjects using trapped ion mobility spectrometry coupled with quadrupole time of flight

Abstract Purpose Proteomic composition of tears is not yet fully characterized and has a great potential to be used as source for discovery of disease‐specific protein biomarkers. The present work is a preliminary evaluation of tear proteome using timsTOF Pro ® , a new highly sensitive mass spectrometry technology. Material and Methods Three tear samples from 3 healthy subjects (HS) and 2 from a dry eye (DE) patient were collected using plastic micropipettes and Schirmer’s strips respectively. After protein elution from the strips, all samples were treated under the same conditions. Proteins were reduced and alkylated then digested in solution with LysC and Trypsin prior to nanoLC‐MS/MS analysis (nanoElute and timsTOF Pro ® , Bruker). Protein identification was performed with Xtandem. Results In total, 1191 proteins were identified in both analytical groups with at least 1 specific peptide: 446 proteins in HS and 1034 proteins in the DE; 802 proteins were exclusively identified in DE (e.g. A2M, HPX, ALDH3A1, PKM, MYH14, ALDH3A1) while 157 were expressed only in controls (e.g. PLOD1, DNAJC3, GOLM1, RNASE4, SDF4, HYOU1). In DE cell structural associated proteins as well as proteins highly correlated with inflammatory and apoptotic processes were detected. Finally, among the proteins that are common to both groups 122 were upregulated in DE vs HS (e.g. ALB, TF, ENO1, ANXA1, GSTP1, S100A9) while 88 were downregulated (e.g. LTF, LCN1, LPO, CST4, LACRT, AZGP1). The upregulated proteins are associated with a blood‐lachrymal barrier failure, inflammatory & immune responses while the downregulated ones sign a lachrymal gland dysfunction. Conclusions Differences in the protein expression between HS and DE arise due to the pathological conditions but also due to the different sampling methods used. This preliminary study could offer a roadmap for designing further studies for the in‐depth characterization of the tear protein profile expression under physiological and pathological conditions.