Protein Profiling of Human Aqueous Humor by Mass Spectrometry

Abstract

Purpose: : We report the first detailed and comparative quantitative analysis of the protein content of aqueous humour from human eyes with cataract by mass spectrometry. In addition to protein profiling, the approach is layered with quantitative proteomics using the iTRAQ methodology.

Methods: : Aqueous humour samples from ten clinically-matched cataract patients were collected, and pairs of patient material were pooled, mixed and divided equally into three aliquots. Proteins from one third of the pair-wise pooled samples were separated by one-dimensional gel electrophoresis, followed by tryptic in situ digestion and LCMS on a hybrid LTQ Orbitrap XL mass spectrometer. Another third was digested in solution and separated by reverse-phased reversed-phase two-dimensional LCMS; and the final third was labelled with 4-plex iTRAQ reagents and separated by 2D-LCMS.

Results: : A total of 198 protein groups were identified across the entire study. Relative protein quantitation with iTRAQ revealed that 69% of the proteins had an approximate equimolar distribution between 3 of the 4 labelled samples, indicating minimal variation between the control cataract samples. The identified proteins were categorised by gene ontology database entries with respect to cellular compartmentalisation, molecular function, and biological processes. One third of the proteins were annotated as extracellular. The major molecular functions of the proteins in aqueous humour are binding and inhibition of proteolytic activity. Complementary to molecular function, the predominant biological processes for the proteins in aqueous humour are assigned to inflammatory and immune response, and transport.

Conclusions: : Our results provide an overview of the proteins present in AH of aged healthy eyes. In summary, the 198 unique protein groups identified via the combined methods are involved in diverse biological processes and function such as binding, inhibition of proteolytic activity, transport, immune, and inflammatory response. This study is an initial step toward establishing the foundations for future proteomic investigations.

Keywords: proteomics • aqueous • cataract