Elsevier

Journal of Proteomics

Volume 75, Issue 13, 16 July 2012, Pages 3877-3885
Journal of Proteomics

In-depth analysis of the human tear proteome

https://doi.org/10.1016/j.jprot.2012.04.053Get rights and content

Abstract

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36 ± 14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.

Graphical abstract

Highlights

► We identify 1543 tear proteins, the largest number of tear proteins reported so far. ► Tears are useful for evaluating cornea, conjunctiva and lacrimal gland function. ► Tear proteome is not a simple reflection of the plasma proteome. ► We provide a reference list of human tear proteome for future biomarker research.

Introduction

The tears, overlying the epithelium of the surface of the eye, are a complex extracellular fluid which is easily and non-invasively accessible for analysis of proteins useful for monitoring both ocular and systemic disease states. The thin (3–40 μm) layer of tears are organized into an outer lipid layer (15–160 nm, non-polar and polar lipid layer with intercalated proteins), middle aqueous phase (proteins, small molecule metabolites, electrolytes, gel-forming mucins, etc.) and inner glycocalyx layer (transmembrane glucoproteins, mucins) which connects the tear film to the epithelial cells of the cornea surface [1]. Several methods of collection of tears are available for varying conditions. The two most popular collection methods are fire polished glass capillary tube and the Schirmer's strip [2], which is particularly useful as it is a standard clinical technique [3], [4], [5].

Efforts to characterize tear composition are continuously growing. A tiny volume of tears (typically 5 to 10 μl under normal condition) has been documented to contain many types molecules, i.e. proteins/peptides, lipids, small molecule metabolites, and electrolytes [6].

de Souza et al. [7] identified 491 proteins in human tears using a comprehensive approach including in-gel/in-solution digestion, LC/MS-LTQ-FT and LC/MS-LTQ-Orbitrap. Our own work routinely detected around 100 tear proteins using online 2D-LC/MS/MS on a QSTAR XL mass spectrometer [3], [5]. Other early studies on human tears only detected fewer than a hundred proteins [8], [6]. Very recently, our group identified 60 small molecule metabolites in normal human tears using an untargeted LC-MS/MS approach on a TripleTOF 5600 mass spectrometer [9]. Combined with previous reports using targeted methods, about 100 different types of small molecule metabolites have been identified in human tears thus far [9]. Tear lipids originating from the oil producing glands in the eyelids, have been studied and more than 150 lipid species from 6 distinct lipid classes were identified in human tears [10].

Tear components both quantitatively and qualitatively reflect the health of the underlying tissues and are useful for their evaluation in health and disease, as well as some systemic conditions. This makes tears a valuable source for objective analysis of abnormal changes due to diseases or other conditions such as corneal wound [11], [12]. Tears have been used as a source for uncovering biomarkers of diseases such as, dry eye [13], [3], [4], [14], blepharitis [15], Sjögren's Syndrome [16], conjunctivochalasis [17], keratoconus [18], [19], autoimmune thyroid eye disease [20], and patients under anti-glaucoma medications [5].

The chemical composition of human tear fluid is probably less complex as compared to plasma and urine but how many proteins tear fluid may contain remains unclear.

In this study, we report the most comprehensive human tear proteome from healthy subjects up to date. This was achieved using offline SCX fractionation followed by nanoRP-LC coupled to a high speed TripleTOF 5600 mass spectrometer.

Section snippets

Chemicals

Acetonitrile (ACN), formic acid (FA), water (LC-MS grade) and ammonium bicarbonate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Protease Inhibitor cocktail was bought from Thermo Scientific (Rockford, IL, USA). Bio-Rad DC protein assay reagents and bovine serum albumin standard were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Trypsin was purchased from AB Sciex (Foster City, CA, USA).

Tear collection and sample preparation

Tear samples were collected from four healthy subjects (3 females and 1 male, average

Human tear proteome

Tear protein samples (total protein amount: 400 μg) were pooled from four healthy individuals (100 μg each). Six fractions were generated using offline SCX fractionation. Each fraction was then analyzed in triplicates by nano-RPLC coupled to a high speed and high resolution TripleTOF 5600 mass spectrometer. The number of unique peptides identified (at 1% FDR) and spectra used in each fraction are given in Fig. 1. The total number of unique peptides/unique proteins identified after combining six

Conclusions

In summary, we have identified 1543 proteins (< 1% FDR) in tear fluid from normal human subjects, which represents the most comprehensive tear proteome to date. This was achieved by using the latest high speed TripleTOF mass spectrometer. The comprehensive tear protein list from this study may be used as a reference list of human tear proteome for biomarker search of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assay for targeted analysis. Tear fluid is an accessible

Acknowledgments

Authors would like to thank Tian Dechao for the assistance in data processing. This work was supported by grant NMRC CG 2010 from the National Medical Research Council (NMRC), Singapore. We also thank Singhealth Foundation's grant in supporting the core proteomics facility.

References (60)

  • L. Tong et al.

    Association of tear proteins with Meibomian gland disease and dry eye symptoms

    Br J Ophthalmol

    (2011)
  • T.T. Wong et al.

    Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication

    Invest Ophthalmol Vis Sci

    (2011)
  • L. Zhou et al.

    Characterisation of human tear proteins using high-resolution mass spectrometry

    Ann Acad Med Singapore

    (2006)
  • G.A. de Souza et al.

    Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

    Genome Biol

    (2006)
  • N. Li et al.

    Characterization of human tear proteome using multiple proteomic analysis techniques

    J Proteome Res

    (2005)
  • L. Chen et al.

    Characterization of the human tear metabolome by LC-MS/MS

    J Proteome Res

    (2011)
  • A.H. Rantamäki et al.

    Human tear fluid lipidome: from composition to function

    PLoS One

    (2011)
  • L. Zhou et al.

    Proteomic analysis of rabbit tear fluid: defensin levels after an experimental corneal wound are correlated to wound closure

    Proteomics

    (2007)
  • L. Zhou et al.

    Analysis of rabbit tear proteins by high-pressure liquid chromatography/electrospray ionization mass spectrometry

    Rapid Commun Mass Spectrom

    (2003)
  • F.H. Grus et al.

    SELDI-TOF-MS proteinchip array profiling of tears from patients with dry eye

    Invest Ophthalmol Vis Sci

    (2005)
  • P. Versura et al.

    Tear proteomics in evaporative dry eye disease

    Eye (Lond)

    (2010)
  • B.S. Koo et al.

    Comparative analysis of the tear protein expression in blepharitis patients using two-dimensional electrophoresis

    J Proteome Res

    (2005)
  • N. Tomosugi et al.

    Diagnostic potential of tear proteomic patterns in Sjogren's syndrome

    J Proteome Res

    (2005)
  • A. Acera et al.

    Changes in tear protein profile in patients with conjunctivochalasis

    Cornea

    (2011)
  • I. Lema et al.

    Proteomic analysis of the tear film in patients with keratoconus

    Mol Vis

    (2010)
  • C. Pannebaker et al.

    Tear proteomics in keratoconus

    Mol Vis

    (2010)
  • R. Okrojek et al.

    Proteomics in autoimmune thyroid eye disease

    Horm Metab Res

    (2009)
  • W. Huang da et al.

    Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources

    Nat Protoc

    (2009)
  • G.L. Andrews et al.

    Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600)

    Anal Chem

    (2011)
  • T. Kocher et al.

    Ultra-high-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides

    Anal Chem

    (2011)
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