Osteoarthritis (OA) is the world's most common age-related joint disease and is characterized by degeneration of cartilage (1, 2). In addition, degradation processes that structurally change the joint are involved in OA, including synovial inflammation, osteophyte formation, and remodeling of subchondral bone (3). This pathology affects up to 70% of the population age >65 years. Cartilage degradation represents a key process during OA pathogenesis. In addition, treatments to manage OA are limited to controlling pain and improving function. Therapies to reduce cartilage degradation and joint destruction are a new challenge in OA treatment. Based on all these comments, methodologies that help us to study in detail the structure of cartilage could improve the knowledge of OA.
Mass spectrometry (MS) facilitates the molecular identification of lipids, peptides, and proteins through the determination of their molecular weight and their fragmentation behavior (4). Proteins from the cartilage and chondrocytes, such as prolargin (PRELP), fibromodulin (FM), aggrecan core protein, decorin, or biglycan, have been previously identified (5, 6). All of them interact with glycosaminoglycans (GAGs) and build the extracellular matrix (ECM) in which the chondrocytes are embedded (6). OA-related proteins, such as cartilage oligomeric matrix protein (COMP) and fibronectin (FN), have been detected by MS and are considered markers of inflammation and tissue remodeling (7). For the first time, the identification and localization (with high spatial resolution) of these 2 proteins have been achieved using the same sample and without any type of labeling.
Matrix-assisted laser desorption ionization–imaging MS (MALDI-IMS) can determine the distribution of hundreds of unknown compounds in a single molecular imaging measurement. MALDI-IMS has been used in the past several years to look for peptides, proteins, and lipids in specific areas of a tissue section with a spatial resolution <50 μm (4, 8, 9). After careful preparation, the tissue section is introduced into the mass spectrometer, and then the proteins, peptides, and lipids are desorbed from discrete pixels from the surface and in an ordered way. Each pixel is linked to the mass spectrum specific for that region. A plot of the intensity of a signal produces a map of the relative abundance of that compound over the imaged tissue (10).
This technology provides a powerful tool for the investigation of biologic processes, as it is not known in advance which segment of the molecular complexity will be revealed. IMS is not necessarily a targeted analytic method, and the technology has been applied to a variety of pathologies, such as neurodegenerative disorders and many cancer types (11, 12). In addition, specific protein patterns revealed by IMS have been shown to be predictive of diagnosis and prognosis (13–15). MALDI-IMS has also been used to detect and map pharmaceutical compounds in sections from dosed tissues (14, 15). Although this is a promising method, there are no previous studies that describe and compare the peptide distribution in human control and OA cartilage. IMS is a new technology that can help us localize and identify the key molecules in OA pathology.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we developed a novel protocol by which to identify and localize peptides/proteins in human control and OA cartilage by MALDI-IMS (17). MALDI-IMS involves the visualization of the spatial distribution of proteins, peptides, or drug compounds within thin slices of samples. It is a promising tool for putative biomarker characterization and drug development. Many investigators have already used MALDI-IMS to localize proteins, peptides, and lipids in diseased tissues (13, 18). For example, MALDI-IMS has revealed novel prognostic markers in Barrett's adenocarcinoma and intestinal gastric cancer, and it has even been used for tumor classification (11, 13, 19–22). However, nothing has been described until now in relation to rheumatic pathologies.
For the first time, we studied the distribution of peptides in healthy and OA human cartilage by MALDI-IMS. Among the proteins in healthy cartilage, we identified unique peptides of aggrecan core protein (m/z 2271.1, m/z 1315.6, m/z 2270.2), decorin (m/z 2763.3), PRELP (m/z 1590.9, m/z 1352.7), and biglycan (m/z 2027.1, m/z 1312.7, m/z 3081.6, m/z 2846.3). We showed an example of the homogeneous distribution of m/z 1590.9 from PRELP and m/z 2278.1 in healthy samples. We saw a similar distribution of PRELP in OA cartilage (data not shown). All these identified proteins interact with GAGs and build the ECM in which the chondrocytes are embedded (23), and they modulate fibril formation of collagen, especially type I collagen, preventing the mineralization of the cartilage (24, 25).
In OA samples, we also identified peptides from aggrecan core protein (m/z 819.4), biglycan (m/z 2027.2, m/z 2846.4), PRELP (m/z 1044.5, m/z 1590.9, m/z 1070.6), collagen α1(II) chain (m/z 2023.9), and protein ELYS (m/z 1025.6), as well as FM (m/z 1955.1, m/z 1361.7, m/z 767.4, m/z 978.5). Under pathologic conditions, FM can activate classical pathways of direct complement binding and plays a role in inflammation of the joint (26).
Regarding the already known OA-related proteins described in the literature, we identified different peptides of COMP in OA cartilage samples. Our results indicate a higher presence of the peptides m/z 2256.1 and m/z 1613.8 in OA versus healthy cartilage. In addition, the distribution in OA cartilage was more abundant in the deep area than in the superficial area (27). Our findings are consistent with the observation that rheumatoid arthritis (RA) patients and OA patients who have high levels of COMP in serum also have bone erosion (28). Skiöldebrand et al demonstrated that galloping horses had high levels of COMP messenger RNA expression and protein in the deep areas of osteochondral fragments from the middle carpal joint (29). Using classic proteomics such as liquid chromatography, FN or COMP can be detected in synovial fluid from controls, but in a lower concentration than in samples from OA patients (30, 31). In our approach, we did not perform any protein separation, identifying the proteins directly from the cartilage tissue.
COMP is an integral structural component of the cartilage matrix that binds to types I, II, and IX collagen and types I and II procollagen. It has been found in high levels in the cartilage, synovial fluid, and plasma of OA and RA patients (32). Only a few studies have looked for the distribution of COMP in OA tissues. By microscopy, COMP has been shown to be present in higher levels in the intercellular matrix than inside the chondrocytes (33, 34), and in rabbits with anterior cruciate ligament transection, it stains positively in the area surrounding apoptotic chondrocytes (35). COMP has been related to endochondral ossification in the developing mouse joint, exhibiting a gradient reduction to the superficial zone (36). The 2 identified peptides are located in the C-terminal end of COMP, which binds types I, II, and IX collagen and regulates fibril formation. Mutations in the C-terminal end exert a dominant-negative effect on both intracellular and extracellular processes. This ultimately affects the morphology and proliferation of growth plate chondrocytes, eventually leading to chondrodysplasia and a reduction in long bone growth. The C-terminal end of COMP is also important because it can directly bind to FN (37). COMP also bound to other proteins associated with OA (FM, CILP, matrix metalloproteinase 3, β6 integrin, and β8 integrin) (further information is available online at http://aigaion.amolf.nl/index.php/publications/show/6021). Thus, using this technique, the identification of peptides in specific areas of a tissue can be directly linked to the presence of the protein (8, 38).
We also identified many peptides from other OA-related proteins like FN. Our results indicate a high presence of the peptides m/z 1401.7, m/z 1593.9, m/z 1431.8, and m/z 1323.7 in OA versus healthy tissues. In addition, all these masses were more abundant in the deep area of the OA cartilage than in the superficial area. By means of Biomap software, we quantified the intensity of the differences in abundance in healthy and OA samples and in superficial and deep areas. We also performed immunohistochemistry studies that validated these results. FN is a glycoprotein that is present at low levels in the ECM of control cartilage, and its increase in OA cartilage produces a change in chondrocyte phenotype and in the activity of metalloproteinases (39). Positive staining for FN in the superficial area of OA cartilage has been described (40); however, the authors pointed out the possibility that the observed FN staining could be due to the penetration of the protein from the synovial fluid into the cartilage.
Our results show high levels of FN in the deep area of OA cartilage, and other investigators have previously shown results in this direction. FN synthesis was measured by radioimmunoassay in bovine articular explants as well as in chondrocytes showing high levels in the deep area (41). Recently, FN was found in the deep area of equine cartilage, and a clear association has been shown between the presence of FN, caspase 3, and apoptosis of chondrocytes (42). It is known that FN fragments from synovial fluid, as well as from OA cartilage, contribute to tissue remodeling, inflammation, and cell death. One hypothesis is that some of the peptides found are related to these FN fragments. However, the antibody used for the immunohistochemistry studies has affinity for the heparin-binding extra domain A segment, and it is known to be specific for cytoplasmic FN and not for FN fragments.
A high abundance of COMP and FN proteins in OA cartilage could be expected. However, we would like to underscore the fact that, to our knowledge, this is the first time these proteins have been detected and identified by MALDI-IMS in human cartilage. In addition, we observed that all of the OA markers detected were increased in the deep area of the cartilage. These results confirm that the study of different parts of the cartilage can give us valuable information for understanding the biologic processes during OA development.
Performing discriminant analysis, we showed that we could differentiate the peptide profiles of OA and control cartilage. Interestingly, the results we obtained in a training data set with 3 healthy cartilage samples and 3 OA cartilage samples were confirmed when we increased the number of samples to 10 donors per group, showing the stability of the technique. Some of the most OA-abundant peaks corresponded to FN, validating the capabilities of the technique even further. One of the OA peptides classified by discriminant analysis corresponded to CILP-1 (m/z 1954.0) (further information is available online at http://aigaion.amolf.nl/index.php/publications/show/6021). Our in-house–developed software localized this mass in the deep area of the cartilage and with a different intensity in OA versus healthy tissue. CILP-1 has been described as an autoantigen in OA pathology and has been said to be regulated by growth factors such as transforming growth factor β (43, 44). In addition, CILP-1 has been found in higher concentrations in the middle and deep areas of aged cartilage as compared with young cartilage (45).
We used the peptide cutter tool from the Swiss-Prot database in peaks that we fragmented but were not identified by the Mascot algorithm. These peaks matched with FN (m/z 1355.7 and m/z 1357.7), α5 integrin (m/z 2376.3, m/z 2377.3, m/z 2378.3), and CILP-2 (m/z 2574.4, m/z 2575.4). Interestingly, different members of the integrin family can bind FN and COMP proteins, demonstrating the close relationship that exists between all these proteins (46, 47). Lorenzo et al found that CILP-2 (C1 isoform) could be used to differentiate OA from RA and nondisease conditions (48). All of the known and unknown OA-related peaks identified by discriminant analysis were also localized by our in-house–developed software in the deep area of the cartilage. Biomap software confirmed this distribution, as we showed for the peptides m/z 2574.4 and m/z 2376.3.
Graphic representation of the intensity versus the number of pixels of peptides with high scores in the first discriminant function revealed that their average intensity was higher in OA samples than in healthy samples. Two different distributions represented the heterogeneity of OA cartilage. This demonstrates the capability of MALDI-IMS to classify different types of tissues according to 1) the score after discriminant analysis and 2) the representation of the intensity versus the number of pixels for a specific mass.
Some of the proteins that were identified by MS/MS were subjected to a protein interaction analysis using String software version 9.0. This resulted in a theoretical OA protein interaction map. All of the included members (COMP, FN, CILP-1, and FM) are connected to different proteins of the integrin family (β5 integrin, β3 integrin, β7 integrin, α5 integrin, α2 integrin, α3 integrin, β8 integrin, β1 integrin, α4 integrin, α2β integrin) (further information is available online at http://aigaion.amolf.nl/index.php/publications/show/6021). Integrins are proteins that connect the cytoskeleton to the ECM. Cell signaling mediated through integrins regulates several chondrocyte functions, including differentiation, matrix remodeling, and cell survival (49). Other investigators have shown that during OA, abnormal integrin expression alters ECM/cell signaling and modifies chondrocyte synthesis, with a subsequent imbalance of destructive cytokines (50). Future experiments to validate the identity of these OA-specific masses could reveal these proteins to be prime species for diagnosis and potential drug targets. These results suggest the potential of MALDI-IMS for the localization and identification of known as well as unknown proteins in the cartilage.
In conclusion, we have simultaneously localized and identified for the first time peptides from human healthy and OA cartilage using MALDI-IMS. The abundance of all the detected OA peptides was higher in the deep area than in the superficial area of the OA cartilage. MALDI-IMS provides a new way to study and image molecules in different areas of the cartilage and to understand molecular signaling pathways and the physiology of OA cartilage in a fast and sensitive way.