Multiplex protein analysis for the study of glaucoma

Abstract

Introduction: Glaucoma, a leading cause of irreversible blindness in the world, is a chronic neurodegenerative disease of multifactorial origin. Extensive research is ongoing to better understand, prevent, and treat progressive degeneration of retinal ganglion cells in glaucoma. While experimental models of glaucoma and postmortem tissues of human donors are analyzed for pathophysiological comprehension and improved treatment of this blinding disease, clinical samples of intraocular biofluids and blood collected from glaucoma patients are analyzed to identify predictive, diagnostic, and prognostic biomarkers. Multiplexing techniques for protein analysis offer a valuable approach for translational glaucoma research.

Areas covered: This review provides an overview of the increasing applications of multiplex protein analysis for glaucoma research and also highlights current research challenges in the field and expected solutions from emerging technological advances.

Expert opinion: Analytical techniques for multiplex analysis of proteins can help uncover neurodegenerative processes for enhanced treatment of glaucoma and can help identify molecular biomarkers for improved clinical testing and monitoring of this complex disease. This evolving field and continuously growing availability of new technologies are expected to broaden the comprehension of this complex neurodegenerative disease and speed up the progress toward new therapeutics and personalized patient care to prevent blindness from glaucoma.

Keywords: Biomarker; glaucoma; immunoassay; mass spectrometry; multiplex protein analysis; neurodegeneration.

Figure 1.

Multifactorial etiological paths of glaucomatous neurodegeneration. Major stressors for the initiation and progression of neurodegeneration in glaucoma, including elevated intraocular pressure (IOP) and aging, along with the genetic/epigenetic susceptibilities, create biomechanical, vascular, and/or immune stress on retinal ganglion cells (RGC). In response to glaucoma-related stress, an interplaying network of pathogenic processes with biomechanical, vascular, metabolic, oxidative, and inflammatory elements promotes RGC degeneration. Apoptosis of RGC somas in the retina, degeneration of RGC axons in the optic nerve, and the loss of retina and brain synapses are regulated by distinct molecular pathways. As much as the individual susceptibility of RGCs to injury, other cell types, mainly including glial cells, also play decisive roles in their demise in glaucoma. Analytical techniques for multiplex analysis of proteins provide a helpful tool for translational glaucoma research.

Figure 2.

Multiple sources of samples utilized in multiplex protein analysis for glaucoma research. Retinal ganglion cells (RGCs), the axons of which make the optic nerve, are specific victims of glaucomatous neurodegeneration. Since the neighboring glia play critical roles in determining the RGC susceptibility to injury, analysis of glial responses is equally important to better understand and treat glaucoma. When considering the heterogeneous cellular composition of the retina and optic nerve tissues and divergent responses of multiple cell types, cell type-specific analysis is crucial. In this regard, isolated samples of RGCs and glia from animal models of glaucoma are increasingly studied to explore pathogenic processes and to develop new treatments. For these studies, RGCs and glial cells are isolated by immunomagnetic cell selection from retina (or optic nerve) lysates. In the retina, microglia migrate but astrocytes are located in the RGC and nerve fiber layers (shown on a histological section of the retina). Besides retina and optic nerve tissues in the posterior segment of the eye, trabecular meshwork, a major site of aqueous humor drainage and intraocular pressure regulation, constitutes an anterior ocular tissue subject to in vitro and in vivo studies of glaucoma. Intraocular biofluids, including aqueous humor and vitreous humor that can be sampled from patients, are also widely studied. As opposed to blood samples, analysis of the secreted molecules in these proximal biofluids can provide more specific information and better guide the personalized patient care on the basis of molecular biomarkers. Protein analysis in the tear film, an extraocular biofluid, may also be useful to assess and monitor ocular surface inflammation that may affect the outcomes of glaucoma surgery. As summarized on this figure, multiplex protein analysis of these samples for glaucoma research employed antibody-based immunoassays, including planar assays (such as multi-analyte ELISAarrays or antibody microarrays) or bead-based assays. In addition, non-antibody-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques used isobaric tags (iTRAQ) or tandem mass tags (TMT) for relative quantification.