Changes in tear protein profile in keratoconus disease

Abstract

Purpose: To analyze tear protein profile variations in patients with keratoconus (KC) and to compare them with those of control subjects.

Subjects and methods: Tears from 12 normal subjects and 12 patients with KC were analyzed by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-mass spectrometry (LC-MS). Analysis of the 2-DE gels was performed using Progenesis SameSpots software (Nonlinear Dynamics). Proteins exhibiting high variation in expression levels (P-value <0.05) were identified using matrix-assisted laser desorption/ionization-TOF spectrometry. For LC-MS analysis, a label-free quantification approach was used. Tears were digested with trypsin, subjected to data-independent acquisition (MS(E)) analysis, and identified proteins were relatively quantified using ProteinLynx Global Server software (Waters).

Results: The 2-DE and LC-MS analyses revealed a significant decrease in the levels of members of the cystatin family and an increase in lipocalin-1 in KC patients. A 1.43-fold decrease was observed for cystatin-S by 2-DE, and 1.69- and 1.56-fold for cystatin-SN and cystatin-SA by LC-MS, respectively. The increase in lipocalin-1 was observed by both methods with fold changes of 1.26 in the 2-DE approach and 1.31 according to LC-MS. Significant protein upregulation was also observed for Ig-κ chain C and Ig J chain proteins by 2-DE. Levels of lipophilin-C, lipophilin-A, and phospholipase A2 were decreased in tears from KC patients according to LC-MS. Serum albumin was found to be increased in KC patients according to LC-MS.

Conclusion: The results show differences in the tear protein profile of KC and control subjects. These changes are indicative of alterations in tear film stability and in interactions with the corneal surface in KC patients.

Figure 1

Flowchart of the experimental approach employed in this study. The left branch summarizes the 2-DE-based analysis. Gray spots represent proteins whose expression level is unaltered in KC samples. Green, blue, and red spots represent differentially expressed proteins, which are identified by MS. The right branch illustrates the LC–MS-based approach. Triangles indicate differentially expressed peptides, whereas circles represent peptides whose expression remains unaltered in KC samples. The colour reproduction of this figure is available at the Eye journal online.

Figure 2

Representative SYPRO Ruby-stained 2-DE gels of tear proteins. Total protein (40 μg) from tear samples was separated on a pH 3–10 immobilized pH gradient strip in the first dimension and by 8–16% SDS-PAGE in the second dimension. (a) 2-DE gel loaded with healthy individual tear samples. (b) 2-DE gel with KC patient tear samples. Arrows illustrate identified protein spots. Cystatin-S (2), Ig-κ chain C region (3, 10), Ig J chain (14, 21), and lipocalin-1 (37).

Figure 3

Principal component analysis of spot volume data. The figure illustrates the two principal components (PC1 and PC2) that explain the majority of variation in the data set, plotted against each other. Each individual gel is displayed by means of filled circles (score plot) in which control gels (red) and KC gels (blue) are indicated. The closer the circles, the more similar the proteomes. Two samples from the control group and one sample from the KC group were excluded because they exhibited a high distribution variation within their respective groups (a, b).