Candidate Serum Biomarkers in Patients with Glaucoma

Abstract

Purpose: : Development of clinically useful biomarkers for glaucoma is an area of active investigation, and proteomics is at the center of these activities. This proteomic study aimed to comparatively identify the presence and abundance of proteins in serum samples of patients with different subgroups of glaucoma and non-glaucomatous controls.

Methods: : Serum samples were collected from 196 patients with glaucoma, including primary open-angle glaucoma (POAG) and pseudoexfoliative glaucoma (XFG), and an age-matched control group of 92 healthy volunteers without glaucoma or any other ocular disease. Un-depleted serum samples were quantitatively analyzed in 5 random samples from each group by two-dimensional capillary liquid chromatography and linear ion trap mass spectrometry (LC-MS/MS). Scaffold Proteome Software was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications assigned by the Protein Prophet algorithm were accepted if they could be established at >99.0% probability and contained at least two identified peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Group differences between the MS/MS data and the ability of the identified serum proteins to discriminate between groups were statistically analyzed using SigmaStat. The Ingenuity Pathways Analysis was used for bioinformatic analysis of biomarkers.

Results: : Analysis of serum protein digests using LC-MS/MS-based proteomic methods identified 325 proteins by two peptides or more at the 0.2% protein and 5.0% peptide false discovery rates. A total of 248 proteins were common to three sample groups (control, POAG, and XFG), while 22 proteins were detected only in glaucomatous samples. As an alternative narrowing down strategy, we also determined whether the serum MS/MS data overlap with our previous high-throughput proteomic data obtained from the glaucomatous human retina or the glaucomatous serum IgG elutes. Overlapping proteins which included various immune or cell death mediators were selected to initially pursue for validation studies in larger number of samples using specific immunoarrays.

Conclusions: : This serum proteomic study in patients with glaucoma and non-glaucomatous controls presented a panel of serum proteins that may serve as candidate protein biomarkers. Ongoing studies in a larger patient population should further validate and establish their potential value for future clinical applications as a diagnostic and/or prognostic tool in glaucoma.

Keywords: proteomics • apoptosis/cell death • neuroprotection