Recherche de peptides biomarqueurs de l’arthrose dans les liquides biologiques

Osteoarthritis is a widespread degenerative joint disease, affecting 10% of people of all ages worldwide and 80% over the age of 75. It is ultimately characterized by an alteration of the entire structure of the joint, including, among other things, progressive degradation of the cartilage as well as inflammation of the synovial membrane (Eveque-Mourroux et al. 2020). To date, there is no treatment for osteoarthritis other than the use of analgesics. In order to better understand the pathophysiology of this disease and to propose therapy, it is essential to have adequate biomarkers allowing the identification of patients presenting risk factors, the early diagnosis of the disease as well as the monitoring of its progression with time. Many biomarkers of this disease have already been described and classified according to BIPED (“Burden of disease, Investigative, Prognostic, Efficacy of intervention, and Diagnostic”) (Felson et al., 2014). They are all quantified by immunoenzymatic methods which have not shown sufficient specificity and sensitivity, discarding their recommendation in clinical routine. The diagnosis of osteoarthritis is essentially carried out by a clinical and radiological assessment. One of the limiting factors of these immunological tests is having an antibody specific for the sought epitope. However, the molecular identification of the epitope is difficult to confirm. Indeed, these biomarkers are peptides which come from the hydrolysis of proteins present in the joint and which can be found in serum, synovial fluid, urine or tissues and more particularly, from type II collagen which makes up 90 to 95% of the cartilage (Birmingham et al., 2007). These biomarkers are molecular structures based on peptide sequences recognized by monoclonal or polyclonal antibodies during the immunoanalysis assay. These markers have largely been proposed following a so-called “top-down” approach during which the sequences of interest for potential biomarkers are conceptualized using knowledge, computerized methods and/or bio-informatics that allows the prediction at a molecular level of the biological effects occurring during the pathology progression (Henrotin et al. 2015 and Baumgartner et al. 2011). The antibody will therefore recognize an epitope in a more or less complex molecular structure. They may or may not be epitopes included in a longer peptide sequence as described by Nemirovsky et al (Nemirovskiy et al., 2007) for TIINE (“Type II neoepitope of sequence GPPGPQG). These authors demonstrated, by liquid phase chromatography coupled with mass spectrometry, several peptides containing the same epitope and consisting of 14 to 47 amino acids. They also showed that it is the 45 amino acid peptide which is the most abundant biomarker. Collagen is a protein that naturally exhibits numerous post-translational modifications (hydroxylation, glycosylation). Thus, it has been detected in various peptide sequences of varying length that certain amino acids (P and K) are hydroxylated and glycosylated. Poole et al., 2004 demonstrated that for the C2C biomarker (EGPPGPQG), the hydroxylation of the Proline in bold was essential for immunogenicity. On the other hand, the hydroxylation of other prolines drastically reduced the antigen (Ag)-antibody (Ab) interaction, which could modify the result of the determination of this biomarker by immunoanalysis. To improve the specificity and sensitivity of these biomarkers, it is essential to determine the molecular structure of these peptides.
This project consists of the development of the dosage of osteoarthritis biomarkers by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) (triple quadrupole type), usable routinely in the laboratory of clinical biology. To do this, we will first develop a protocol for the identification and validation of osteoarthritis biomarkers (degradation products of collagen II) by high-resolution LC-MS/MS and using a cohort of osteoarthritis patients whose clinical and radiological data will be collected. The use of LC-MS/MS will allow us to overcome possible variations in affinity between Ag and Ab during immunological assays as described by Poole et al and to increase the specificity and sensitivity of the quantification. This identification and validation protocol could in the future be applied to propose the multiplex, specific and sensitive dosage of biomarkers exploring the BIPED classification.

Kamphorst, J. J., van der Heijden R., DeGroot J., Lafeber F. P. J. G., Reijmers T. H., van El B., Tjaden U. R., van der Greef J., and Hankemeier T. 2007. “Profiling of Endogenous Peptides in Human Synovial Fluid by NanoLC-MS: Method Validation and Peptide Identification.” Journal of Proteome Research 6 (11): 4388–96.

Bauer A.D.C, Hunter D.J., Abramson S.B., Attur M., Corr M., Felson D., Heinegård D., Jordan J.M., Kepler T.B.,  Lane N.E., Saxne T., Tyree B., Kraus V.B.2006. Review Classification of osteoarthritis biomarkers: a proposed approach1. Osteoarthritis and Cartilage 14 (8): 723-727.

Hellio Le Graverand,M.P., Brandt K.D., Mazzuca S.A., Katz B.P., Buck R., Lane K.A., Pickering E., Nemirovskiy O.V., Sunyer T.,  Welsch D.J. 2006. Association between concentrations of urinary type II collagen neoepitope (uTIINE) and joint space narrowing in patients with knee osteoarthritis.  OsteoArthritis and Cartilage 14: 1189e1195.

L’Institut de Chimie Physique (ICP, UMR 8000) est une unité mixte de recherche interdisciplinaire ancrée en Chimie, avec des projets aux interfaces avec la Physique et les Sciences du Vivant. L’Unité est structurée autour de quatre groupes scientifiques (CAPRI, CpSysBio, TEMiC, TheoSim).

Le doctorant sera accueilli au sein de l'équipe CAPRI/LETIAM, plus particulièrement spécialisée dans l'analyse des mélanges complexes biologiques, par le couplage de la chromatographie liquide à la spectrométrie de masse. Ce travail s'effectuera en collaboration avec l'hôpital Cochin de Paris, l'unité INSERM 1124 (HealthFex), la plateforme protéomique Necker (SFR Necker) et l'UMS IPSIT (Inserm (US 31), CNRS (UMS 3679), Paris Saclay).

L’étudiant (e) , biochimiste ou chimiste ayant des connaissances sur les biomolécules, intéressé(e) par le projet devra avoir une connaissance théorique des couplages de la chromatographie avec la spectrométrie de masse dans un contexte d’analyse protéomique/peptidomique. Il devra présenter une appétence pour les applications cliniques.