Proteomic signature of the murine intervertebral disc

Abstract

Low back pain is the most common musculoskeletal problem and the single most common cause of disability, often attributed to degeneration of the intervertebral disc. Lack of effective treatment is directly related to our limited understanding of the pathways responsible for maintaining disc health. While transcriptional analysis has permitted initial insights into the biology of the intervertebral disc, complete proteomic characterization is required. We therefore employed liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) protein/peptide separation and mass spectrometric analyses to characterize the protein content of intervertebral discs from skeletally mature wild-type mice. A total of 1360 proteins were identified and categorized using PANTHER. Identified proteins were primarily intracellular/plasma membrane (35%), organelle (30%), macromolecular complex (10%), extracellular region (9%). Molecular function categorization resulted in three distinct categories: catalytic activity (33%), binding (molecule interactions) (29%), and structural activity (13%). To validate our list, we confirmed the presence of 14 of 20 previously identified IVD-associated markers, including matrix proteins, transcriptional regulators, and secreted proteins. Immunohistochemical analysis confirmed distinct localization patterns of select protein with the intervertebral disc. Characterization of the protein composition of healthy intervertebral disc tissue is an important first step in identifying cellular processes and pathways disrupted during aging or disease progression.

Fig 1. Proteomic signature of the murine intervertebral disc.

Pie chart depicting gene ontology (GO) analysis of proteins identified in the murine intervertebral disc. Representation of the distribution of proteins according to their molecular function as (A) biological process, (B) molecular function, (C) cellular component, and (D) protein class. Categorizations were based on information provided by the PANTHER classification system (www.pantherdb.org; v9.0). A total of 10 groups for molecular function, 12 groups for biological process, 7 cellular components, and 27 protein classes were detected. Further analysis of the intracellular classification revealed that the majority of proteins were characterized as cytoskeletal, followed by cytoplasmic, organelle and protein-transport ATP synthase complex.

Fig 2. Immunohistochemical staining validating the localization of proteins in the murine intervertebral disc.

Representative images demonstrating the immunolocalization of NFAT5, versican, Sox9, and BSP within the murine IVD. Each protein demonstrates a distinct pattern of localization within specific compartments of the disc. NFAT5 is localized to the NP and CEP, versican is present throughout the disc with high levels detected in the outer annulus, Sox9 is present in NP and CEP and BSP is only detected in the CEP. For each protein-specific antibody, sections corresponding to 3 IVDs were assessed for each animal; n = 3 mice. Scale bar = 100 μm.