Quantitative proteome analysis of normal and degenerated intervertebral disc tissue

Introduction:

Intervertebral disc degeneration (IVDD) is a musculoskeletal disorder affecting people of all ages and causes low back pain. Factors causing IVDD includes genetics, trauma, ageing, life style, etc. resulting in excess catabolic over the anabolic responses. IVDD is also associated with modic changes as seen in MRI including inflammation and edema. Understanding the pathophysiology is crucial to manage the disease conditions. The present study explores the quantitative proteome analysis of normal and degenerated disc tissue to detect the differentially expressed proteins.

Methods:

Intervertebral disc tissues were obtained from brain-dead organ donors (TRANSTAN-214/2016; Transplant Authority Government of Tamil Nadu, India) and patients undergoing surgery with written informed consent. The study subjects include control, modic and non-modic intervertebral disc tissue. Proteins were extracted with 8M urea in 50mM TEAB buffer, sonicated for 5 cycles with 15 sec pulse on/off and isolated by centrifugation. After reduction and alkylation, proteins were digested with trypsin, labeled with TMT-6 plex kit and subjected to MS/MS acquisition in Q-Exactive plus Orbitrap quadrupole mass spectrometer. The experiments were performed as four independent experiments with three technical replicates to identify the differentially expressed proteins. Using Proteome Discoverer 2.5, the protein abundance were detected for further bioinformatic analysis.

Results:

Using mass spectrometry, 1200 proteins were identified in two out of four experiments (50% cut-off). Our earlier study using label free mass spectrometry revealed the upregulation of complement proteins and downregulation of ECM proteins in degenerated disc compared to normal disc (Rajasekaran et al., 2019). In this study, we specifically determined the expression of various complements and ECM related proteins from the TMT mass spectrometry data. In the complement pathway, complement proteins C3, C5 and MAC proteins (C6-9) were upregulated in modic and non-modic tissue. Interestingly, complement regulators including CFH and CFI were increased in modic phenotypes. In ECM-receptor interaction pathway, CHAD, THBS3, TNXB were downregulated in modic and non-modic phenotypes compared to control. While the expression of collagens such as COL2A1, COL6A1, COL6A2 were decreased in modic.

Conclusion

The present study revealed the alteration of complement and ECM proteins in degenerated disc compared to control. Although modic and non-modic condition activates complement pathway, the distinct expression of complement regulators CFH and CFI in modic, indicating the activation of alternate complement pathway. This findings hypothesize that bacterial infection triggers the alternate complement pathway and the infection leads to the downregulation of matrix proteins and collagens in modic. Further, the results should be validated in future by targeted PRM based mass spectrometry.