Ligamental Proteomic Profiling Of Patients With Ossification Of Cervical Posterior Longitudinal Ligament

Objective Ossification of the posterior longitudinal ligament (OPLL) is considered as enthesopathy or as inflammation of the tendinous and ligamentous attachments to the bone. The incidence of OPLL in Aisa old people could be 20%-34%. Hormonal and metabolic factors or hereditary factors have been proposed to involved during pathologic ligamentous ossification of the OPLL. However, the mode of inheritance and the etiology for OPLL are still obscure. There are still no definitive biomarks of OPLL that might be used to achieve an early diagnosis. To find an easier and simpler diagnostic method, to find the pathogenic proteins of OPLL, and provide evidence for pharmacological research for OPLL, we analyzed cervial posterior longitudinal ligament for the alternation in their proteomes.Methods Cervial posterior longitudinal ligaments were collected from 5 OPLL patinets and 5 normal subjects without OPLL. Proteins lysates were extracted after the ligaments were disrupted by abrading and ultrasonic. The internal standard is created by pooling an aliquot of all samples and labelling it with CyDye DIGE fiuors Cy2. Protein lysate of OPLL Samples and noraml samples were labeled randomly with CyDye DIGE Fiuors Cy3 or Cy5. The labeled samples together with internal standard were seperated using two-dimensional gel electrophoresis (2-DE). Gels were scanned and analyzed by Typhoon Variable Mode Imagers. The protein spots which had change in intensity were marked, and picked by hand in another gel. Digested peptides were identified by MALDI-TOF MS, MALDI-TOF-TOF MS or LC/MS. Literature review was performed to understand the function, localization of special protein or peptide. Real time-polymerase chain reaction (RT-PCR) had also been done to validate the results of differential gel electrophoresis.Results Fifty protein spots were detected that differentailly expressed between OPLL samples and normal samples. Forty-five of them were picked up, of which twenty-eight proteins or peptides were identified. Differential expression of 6 proteins, including carbonic anhydrase I, NAD(P) dependent steroid dehydrogenase-like, osteoglycin OG, alpha-1 collagen VI, biliverdin reductase B, and nebulin-related anchoring protein, was examined by RT-PCR tests, and 3 of them was validated.Conclusions Methods developed by our research for sample preparation, proteomic profiling, protein identification, and validation uncovered several differentially expressed proteins for OPLL. Baesd on the study result, we now conclude that 6 proteins, alone or in combination, were putative disease biomarkers. One of the imitations faced was the relatively small number of samples with sufficient protein contents to be investigated by 2-DE in combination with protein identification by MS.