Quantitative Proteomic Analysis of Human Aqueous Humor by an Amine-Specific Peptide-Based Labeling Technology

Abstract

Purpose: : Characterizing protein expression in ocular fluids like aqueous humor (AH) is a key objective towards understanding pathomechanisms of eye disorders. Proteomic technologies applied to AH offers the potential of detecting large numbers of proteins. To identify factors that are specific for a disease, expression levels of proteins need to be compared between healthy and diseased samples. The "isobaric tagging for relative and absolute quantification" (iTRAQ) technique is an amine-specific peptide-based labeling technology for the simultaneous identification and quantitation of biological samples.The goal of this study was to assess the feasibility of labeling AH samples with iTRAQ for relative quantification of protein levels.

Methods: : AH samples were obtained from patients undergoing cataract surgery without any other eye disease. 4 samples were labeled with 4-plex iTRAQ reporter reagents and separated by 2-dimensional liquid chromatography mass spectrometry (2D-LCMS) on an Orbitrap mass spectrometer. For the final relative quantitation of a protein, the median ratio of the relative intensities of the reporter ions of all spectra from specific peptides for each protein is used.

Results: : Compared to the iTRAQ reporter ion at m/z 114.1112, 68.75% of all the proteins identified had a maximum fold change of 1.5 in the 115 or 116 channels. One example of the quantitative information of a single protein is shown with SERPINA1: Relative to m/z 114.1112, the ratio of SERPINA1 in the 4 samples chosen for quantitative analysis is 1.12±0.08 to 1.26±0.17 to 0.56±0.23, indicating that there was minimal variation in the protein quantity in three of the samples.

Conclusions: : We have shown for the first time that it is feasible to compare the proteome of individual AH samples with this methodology. The analytical approach described here will be particularly beneficial in tracking protein changes during individual patient therapy over a time period and to gain new pathomechanistic insights.

Keywords: proteomics • aqueous • protein purification and characterization