Analysis of the Chemical Composition of Tears Based on Various Samples

The tears, covering the epithelium of the exterior of the eye, are an intricate extracellular fluid which is easily and noninvasively available for examination of proteins for monitoring both ophthalmic and systemic disease conditions. The tiny (3–40 μm) coating of tears are arranged into an external lipid layer (15–160 nm, non-polar and polar lipid layer with intercalated proteins), middle aqueous phase (small molecule metabolites, proteins, electrolytes, gel-forming mucins, etc.) and internal glycocalyx film (contains transmembrane glycoproteins and mucins) which joins the tear film to the corneal surface epithelial cells (Green-Church et al., 2011).

Several means of collection of tear samples are available for variable circumstances. The two most common collection approaches are fire polished glass capillary tube and the Schirmer's strip (Green-Church et al., 2008), which is mainly suitable as it is a typical clinical technique (Zhou et al., 2009, Tong et al., 2011, Wong et al., 2011). A minute volume of tears has been found to contain several types of molecules, i.e. proteins, lipids, glycoproteins, small molecule metabolites, and electrolytes (Zhou et al.,2006). De Souza et al. recognized 491 proteins in human tear sample using in-gel/in-solution digestion, LC/MS-LTQ-FT and LC/MS-LTQ-Orbitrap method. The work routinely detected around 100 tear proteins using online 2D-LC/MS/MS on a QSTAR-XL mass spectrometer (Zhou et al.2009., Wong et al.,2011). Other early studies on human tears only detected fewer than a hundred proteins (Li et al., 2005). Zhou et al. identified 60 small molecule metabolites in tears of healthy human using LC-MS/MS on a Triple-ToF 5600 mass spectrometer (Chen et al.,2011).

Combined with earlier reports using targeted approaches, about 100 different kinds of small molecule metabolites have been recognized in human tears thus far (Chen et al.,2011). Tear lipids generated from the oil manufacturing glands present in the eyelids, have been examined and approximately 150 lipid species from 6 different lipid classes were identified in healthy human tears (Rantamäki et al., 2011).

Tear constituents both quantitatively and qualitatively shows the health of the ocular tissues and are pivotal in assessment in pathophysiology and some systemic conditions. Thus, tears a much-appreciated source for the analysis of abnormal fluctuations due to ocular complications like corneal wound (Zhou et al.,2007, Zhou et al., 2003). This makes tears as a valuable source for discovering new biomarkers of diseases such as, blepharitis (Koo et al., 2005), dry eye (Grus et al.,2005, Versura et al.,2010), keratoconus (Lema et al.,2010, Pannebaker et al.,2010), autoimmune thyroid eye disease (Okrojek et al.,2009), Sjögren's Syndrome (Tomosugi et al., 2005), and patients under anti-glaucoma medications (Wong et al.,2011). The constituents of human tear fluid are comparatively less complex than plasma and urine but how many proteins in tear fluid may appear during any pathogenic conditions remains unclear.

Proteomic classification of human serum for documentation of disease-specific biomarkers promises to be a commanding prognostic means for detection of the onset, development and progression of human diseases (Anderson et al.,2002) Serum offers a rich sample for diagnosis since it has because of the condition specific expression and release of proteomic biomarkers into the bloodstream in response to specific physiological circumstances.

The significant portion of serum is constituted by albumin approximately 10 or more. So, before going for any proteomic analysis the crude serum sample must be separated from the scarcest of proteins. In addition, numerous serum proteins are alike in molecular weight and isoelectric point, making protein separation problematic. Therefore, condition specific biomarkers for any disease, that may be present at very minute concentrations in serum, could be masked by more copious proteins having identical biophysical characteristics (Hanash et al., 2003). Albumin constitutes approximately 55% to 75% of the total protein in serum content. Even after albumin removal, serum remains a complex protein mixture of five other high-abundant proteins IgG, IgA, haptoglobin, transferrin and antitrypsin. Together, these six most abundant proteins constitute over 85% of the total human serum proteome (Zhang et al.,2003).

Generally, Cibracon Blue (Gianazza et al.,1982) and protein A/G chromatography techniques (Hage et al.,1999) have been used for depletion of albumin and the immunoglobulins. However, an increasing number of procedures for the elimination of other high-abundant serum proteins are becoming commercially available, making serum proteomic analysis a more routine laboratory procedure (Zhang et al.,2003).

Albumin is a carrier/transport protein that binds other important entities in blood (Burtis et al., 2001); and consequently, the removal of albumin from a serum sample could also remove other physiologically important species. Following major fraction of protein depletion, Coomassie blue stained 2-dimensional electrophoresis (2-DE) gels reveal less number of protein spots compared to crude serum. Silver staining of the protein-depleted sample is generally performed to reveal more spots since it is sensitive to even very low concentration of protein. Chan and coworkers (Wang et al., 2003) employed an affinity spin tube filter technique to eliminate albumin and IgG to enrich the visible low-abundant cancer biomarkers in serum. Steel and co-workers (Steel et al.,2003) employed an immuno-affinity resin to eliminate albumin and IgG from healthy human serum samples to get a better resolution for proteomic analysis.

These examples relied on traditional 2-DE, and while 2-DE is limited in the ability to detect low abundant proteins and it remains a significant method for the separating a complex mixture of proteins (Pieper et al.,2003). Notably, the introduction of fluorescent dyes for 2-DE analysis (Patton et al.,2000, Patton et al.,2001) has offered many improvements over conventional silver staining and Coomassie blue staining techniques.

For example, fluorescent stains like SYPRO ruby, Deep purple, and Lightning Fast (Mackintosh et al.,2003) which are particularly used for post-electrophoretic staining. Which bind non-covalently and deliver a broader dynamic range of protein detection. 2-D differential gel electrophoresis (DIGE) has been developed for multiplex proteomic analysis based on the spectrally resolvable fluorescent dyes Cy2, Cy3, and Cy5.15 The Cy dyes covalently attach to proteins via lysine residues before electrophoresis. Three proteomic samples can be electrophoresed together on the same gel lessening the complications of gel-to-gel evaluations.

This multiplex capability improves the consistency of comparative tests by increasing the statistical importance of variable expression. In addition, the Cy2 dye can be used to label a pooled, internal standard permitting more reliable and accurate gel-to-gel evaluations in larger sample sets.15 There have been several examples of the application of 2-D DIGE method to distinguish proteomic differences (Kernec et al.,2001, Zhou et al.,2002, Yan et al., 2002, Ruepp et al., 2002, Lee et al., 2003, Hu et al., 2003, Van den Bergh et al., 2003, Alaban et al., 2003). In the study presented here, 2-D gel electrophoresis was utilized to characterize human serum after removal of two abundant serum proteins. The serum, before and after depletion of high-abundant proteins, was analyzed by SDS-PAGE as a preliminary screen for protein removal efficacy.

Finally, 2-D gel electrophoresis was used to examine serum, after successful removal of high-abundant proteins, defining over 255 protein spots. This result represents a significant improvement in proteomic characterization of human serum.

In this study, we also reported the human tear and serum proteome of normal (NOM) healthy, diabetic (DM) and diabetic retinopathy (DR) affected individuals for differential proteomic analysis. This was achieved using 2D gel electrophoresis followed by ESI-MS coupled to a high-speed Triplet ToF 5000 mass spectrometer.