Multidimensional Protein Identification Technology

Abstract

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in antioxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states.The aqueous humor (AH) is a clear fluid that fills the anterior segment of the eye and bathes the lens, iris, and corneal endothelium [1]. It is secreted by the ciliary body and functions to provide nutrients and remove waste from avascular tissues [2], as well as create the intraocular pressure that maintains the convex shape of the cornea. The AH has antioxidant properties and purported immune response roles during inflammation and infection [3,4]. However, the proteins that are responsible for carrying out these functions are largely unknown. The protein content of the AH has been studied extensively [5][6][7][8][9][10][11][12]; however, due to limitations in technology, an extensive and definitive description of human AH proteins has yet to be provided. Furthermore, proteins in the AH are thought to be involved in development of several eye diseases [13,14], and investigating the AH proteome will facilitate generation of new hypotheses regarding the etiology of such pathologies. Thus our goal in this study was to investigate the AH proteome and determine its protein constituents with high confidence using an advanced proteomic approach.