A high definition proteome of the human intervertebral disk: understanding lower back pain

Introduction:
Chronic lower back pain is an extremely common musculoskeletal condition that severely impacts quality of life and frequently leads to disability. Indeed, it is the highest ranking musculoskeletal disorder as measured by years lived with disability worldwide [1]. One of its leading causes is the degeneration of the lumbar intervertebral disk [2]. While both pharmaceutical and surgical options are available for symptomatic treatment, there is no clear understanding of the cause(s) of the condition nor its pathogenesis, partially due to the lack of suitable disease models. Global proteomics has been used to interrogate human samples from both intervertebral-healthy and -degenerate donors, however met with limited success due to limitations in sampling and analytical methodology [3]. In the light of recent advances in tissue proteomics [4], we have applied DIA-MS analysis to defined regions of lumbar disks from intervertebral-healthy donors to generate a high definition spatially resolved proteome as a basis to study intervertebral disc degeneration in patient samples.
Methods:
Disk tissue from intervertebral-healthy donors was washed to remove blood, then sectioned to represent different functional regions. Samples were lysed by pressure cycling in a Barocycler 2320XT (PBI), digestion with trypsin using an optimized SP3 protocol in a Kingfisher Duo Prime (Thermo Fisher Scientific) and analysed by 120 min diaPASEF acquisitions in a timsTOF Pro 2 mass spectrometer (Bruker). Data were analysed by directDIA using Spectronaut v18.5 (Biognosys) against the human UniProtKB reference proteome v08/23. Differentially abundant proteins were analysed for functional enrichment using the ShinyGO v0.80 tool (SD State University).
Results:
Proteomic profiling of samples (n=4) from the Nucleus pulposus (NP) and the Annulus fibrosus (AF) as well as from adjacent regions of the disk led to detection and quantitation of a total of 8721 protein groups evidence by 153856 precursors, all at 1% FDR, at good quantitative reproducibility, with in-group CVs of 23.8-34.3%. Differential abundance analysis showed highly regionalized proteomes, with a strong enrichment of extracellular matrix (ECM), granule- and vesicle- associated proteins in NF, and of proteins representing cellular organization (ribosomal, mitochondrial, focal adhesion) in AF. The results show that deep spatial proteomics of the intervertebral disk is readily possible, and can be utilized to study processes of degeneration. We plan to extend our approach to analysis of the glycoproteome in the future.
Literature:
[1] Hoy et al., PMID: 24665116; [2] Kirnaz et al., PMID: 34929784; [3] Rajasekaran et al., PMID: 32973250; [4] Neuenroth et al., bioRxiv (2024).