Comprehensive Characterization of Human Tear Proteome Using Nano–Liquid Chromatography–QTOF Tandem Mass Spectrometry and Quantitative Proteomics (iTRAQ)

Abstract

Purpose: : Proteomics provides a comprehensive approach for cataloging the components of the tear proteome which can be used to understand and diagnose a variety of ocular surface diseases. In this study, an alternative proteomics strategy was developed to explore the human tear proteome and carry out quantitative proteomics (iTRAQ) to search for biomarkers for dry eye.

Methods: : Tear samples obtained from five normal human subjects (male, average age: 31) were eluted with PBS from Schirmer strips. Tears were pooled and an aliquot (equivalent to 150 µg total protein) was used in the analysis. Reverse phase high pressure liquid chromatograph (RP–HPLC) was used as the first step to separate intact proteins into 30 fractions (3 minutes per fraction). Each fraction underwent tryptic digestion was analyzed by nanoLC–nano–ESI–MS/MS to characterize protein components in each fraction. To reveal dry eye associated proteins, we used multiplex labeling reagent (iTRAQ) with multidimensional nanoLC–nano–ESI–MS/MS to analyze tears from patients with dry eye compared with tears from normal controls.

Results: : Using this proteomic approach, we identified over 80 unique proteins with high confidence (>95%). Among them, 41 proteins matched with the NEIBank lacrimal gland database and 33 proteins were found in the HUPO Plasma Proteome Project database. iTRAQ quantitative results (relative to normal controls) showed that S100 A8 (2–fold), S100 A9 (2–fold), alpha–enolase (2.5–fold) and alpha–1–acid–glycoprotein 1 (10–fold) were up–regulated in tears of patients with dry eye, whereas, lactotransferrin (1.5–fold), lysozyme (1.8–fold), prolactin–inducible protein (2–fold) and antileukoproteinase 1 (1.5–fold) were down–regulated in tears of patients with dry eye.

Conclusions: : Qualitative and quantitative proteomic techniques expand the coverage of the human tear proteome to search for biomarkers associated with eye diseases. Rather than a "shotgun" approach, the advantages of this separation of intact proteins using HPLC followed by analyzing the tryptic digests with nanoLC–nano–ESI–MS/MS strategy include reducing the complexity of tear protein mixture and tolerance for a wide dynamic range of protein levels.

Keywords: proteomics • cornea: tears/tear film/dry eye • lacrimal gland