Background/Purpose:

In this study we have identified proteins differentially abundant in the serum of Osteoarthritis (OA) patients comparing four different progressive pathological grades using mass spectrometry and iTRAQ technique for relative quantification. Our final aim is to establish a panel of biomarkers useful to predict the pathology in pre-radiological stages (early Osteoarthritis biomarkers), but also for its handling and the developing of trials of treatment in radiological stages (late Osteoarthritis biomarkers).

Methods:

15 individual samples for each condition (OA Grade 0, pre-radiological stage Grade I, and radiological stage grades II-III and IV) were pooled in three groups with the aim of reducing the contribution of individual extreme values. After enrichment in the low-abundant protein fraction, the pooled samples were subjected to in-solution digestion, followed by iTRAQ labelling following manufacturer instructions and Reverse Phase peptide separation in a LC system (Agilent 1200).  Fractions were again separated in a nanoLC system (Tempo, Eksigent) automatically deposited on a MALDI plate and analyzed by MSMS in a 4800 MALDI-TOF/TOF system (ABSciex). Relative quantitative analysis was done using ProteinPilot software (ABSciex) and modulated proteins were analyzed with String 9.0 software.

Results:

We have detected two big sets of serum proteins modulated in the early pre-radiological OA process. Serum levels of apolipoproteins are altered when comparing Grade I vs. Grade 0. Specifically, apolipoprotein E and apolipoprotein B-100, that mediates the binding, internalization, and catabolism of lipoprotein particles are accumulated in Grade I samples. Furthermore, up to six components of the complement, a group of proteins involved in immune response and inflammation are decreased in serum in early OA grades. Among them, complement component 5 -C5-, that have been recently identified as key player of the OA process, is much less abundant in any OA grade in comparison to Grade 0. Proteins previously described as putative biomarkers by our group and others, like histidine-rich glycoprotein, gelsolin and decorin, are also modulated in our study, but at later radiological stages -Grade II/III and Grade IV vs. Grade I and Grade 0-.

Conclusion:

Our results indicate that early pathological grades of the OA process are linked to an imbalance in the metabolism and, specifically, in the lipid metabolism. Altered serum levels of apo-lipoproteins could be used, in combination with other “dry” biomarkers, as an indicator for early OA process and to detect the pathology in pre-radiological stages. Furthermore, the lower serum levels in the OA grades detected for the complement component 5 -C5-, strongly support recent in vivo data indicating that a decrease of this protein is linked to the development of the disease.

Table 1: Summary of the results.