An In-Depth View of the Porcine Trabecular Meshwork Proteome

Abstract

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

Keywords: Sus scrofa; glaucoma; mass spectrometry; model organism; proteomics; trabecular meshwork.

Figure 1

Processed porcine trabecular meshwork (TM). (A) TM sample acquisition is difficult due to small size of processed TM tissue as indicated by the coin comparison. (B) Isolated TM displays no connective tissue remains indicating the purity of TM tissue sample (5× magnification). TM sample shows a typical mesh-like structure ((C) 10× magnification, (D) 40× magnification), (E) TM corresponding exemplary extracts fractionated by 1D-SDS PAGE using three different lysis protocols (LB1 = 2 M thiourea, 7 M urea, 30 mM tris-HCl, 4% CHAPS, 2% ASB-14 (8 µg protein); LB2 = 50 mM tris-HCl, 8 mM Na₂HPO₄, 137 mM NaCl, 3 mM KCl, 0,5% SDS, 0,5% triton X-100, 0,5% DDM (8 µg protein); LB3 = 0.5% DDM (50 µg)); all different extracts were used for combinatory BULCMS TM proteome analysis.

Figure 2

The majority of the porcine trabecular meshwork (TM) could be identified by use of the PEAKS combinatory de novo peptide sequencing/database related protein identification strategy. In summary, more 3747 proteins could be documented for the porcine TM.

Figure 3

(A). The majority of identified proteins correspond to the intracellular fraction of the trabecular meshwork (TM) proteome inferred from gene ontology (GO) annotation analysis. (B) Exclusively extracellular proteins (Ex) as well as proteins with localization in the extra-and intracellular milieu (Ex/In) correspond predominantly to extracellular vesicles indicating secretory processes. (C) Most intracellular TM proteins correspond to the nucleus followed by the cellular plasma membrane. (D) The majority of “neuronal associated proteins” are intracellular protein species.

Figure 4

Exemplary identification of a prominent barrier function-related protein of neuronal relevance in the porcine trabecular meshwork (TM) by use of the established BULCMS workflow. A unique reporter peptide peak 848.42 m/z (MS) was fragmented by collision-induced decay (CID) (MS/MS) leading to the stringent identification of neuroblast differentiation-associated protein AHNAK (FDR < 1%) and was supported by both, database-related and de novo peptide sequencing analysis. (AHNAK image from the RCSB PDB (rcsb.org) of PDB ID 4FTG [22]).