Discovery and characterization of irregularly sulfated chondroitin/dermatan sulfate domains and their interactions by ion mobility mass spectrometry

Project Number: PN-III-P4-ID-PCE-2020-0209

Project Director: CS 1 Prof. Dr. Fiz. Alina-Diana Zamfir

Project Title:Discovery and characterization of irregularly sulfated chondroitin/dermatan sulfate domains and their interactions by ion mobility mass spectrometry (Acronym: CHON-DERM-IMS)

Discovery and characterization of irregularly sulfated chondroitin/dermatan sulfate domains and their interactions by ion mobility mass spectrometry
Scientific Report

Proteoglycans are the fundamental components of the extracellular matrix and represent a special category of proteins, highly glycosylated, consisting of a protein core covalently linked to one or more linear, heterogeneous and sulfated polysaccharides known as glycosaminoglycans. The biological functions of proteoglycans are determined by the structure of the glycosaminoglycan chains in their constitution. Among glycosaminoglycans, chondroitin sulfate (CS) and dermatan sulfate (DS) are found in a variety of proteoglycans, being predominantly expressed in mammalian tissue where they are responsible for fundamental events and various pathologies, including malignant transformations. Since in CS/DS chains sulfation is not regulated, undersulfated and oversulfated species appear in nature and play major biological roles. Therefore, the functions of CS/DS and of the proteoglycans that contain them are influenced by the sulfation code, i.e. the number of sulfate groups and their positions. In recent years, worldwide research in the field has been oriented towards the development of the most efficient analytical methods, capable of providing precise information on this type of biomolecules. In this international context, in the CHON-DERM-IMS project a new and robust methodology based on the modern ion mobility mass spectrometry (IMS MS) technique was developed for the analysis of irregularly sulfated CS/DS domains and their interactions. The research carried out within the project led to: a) the optimization and introduction for the first time in the world of the IMS MS method in the research of complex hybrid CS/DS chains; b) the first application of IMS MS to the study of CS/DS species expressed in the human brain; c) the discovery of new CS/DS sequences in the human extracellular matrix; d) detection and structural investigation of under- and oversulfated CS/DS motifs in various proteoglycans, including those expressed in the central nervous system; e) identification of new biologically active CS/DS sequences based on their functional interactions. The obtained results demonstrated that IMS MS is a versatile and integrated technique, which allows a fast, total and ultra-sensitive analysis, hence it is expected that the protocols developed within CHON-DERM-IMS will find wide applicability for studies related to other types of biomolecules, especially biomarkers of severe diseases.

Proteoglicanii sunt componentele fundamentale ale matricii extracelulare si reprezinta o categorie speciala de proteine, puternic glicozilate, formate dintr-un miez proteic legat covalent de una sau mai multe polizaharide liniare, heterogene și sulfatate cunoscute sub numele de glicozaminoglicani. Functiile biologice ale proteoglicanilor sunt determinate de structura lanturilor de glicozaminoglicani din constitutia lor. Printre glicozaminoglicani, condroitin sulfatul (CS) și dermatan sulfatul (DS) se găsesc intr-o varietate de proteoglicani, fiind exprimati cu precadere în țesutul mamiferelor unde sunt responsabili pentru evenimente fundamentale și diferite patologii, inclusiv transformările maligne. Intrucat în lanturile de CS/DS sulfatarea nu este regulata, in natura apar specii subsulfatate și suprasulfatate care joacă roluri biologice majore. Prin urmare, funcțiile CS/DS si ale proteoglicanilor care ii contin sunt influențate de codul de sulfatare, adică de numărul de grupări sulfat și pozitiile lor. Din acest motiv, in ultimii ani cercetarile in domeniu de la nivel mondial au fost orientate inspre dezvoltarea unor metode analitice cat mai performante, capabile sa ofere informatii precise asupra acestui tip de biomolecule. In acest context de la nivel international, in proiectul CHON-DERM-IMS a fost dezvoltata o metodologie noua si robusta bazată pe tehnica ultamoderna de spectrometrie de masă cu mobilitate ionica (IMS MS) pentru analiza domeniilor neregulat sulfatate de CS/DS și a interacțiunilor acestora. Cercetarea efectuata in cadrul proiectului a condus la: a) optimizarea si introducerea pentru prima dată in lume a metodei IMS MS în cercetarea lanturilor complexe hibride de CS/DS; b) prima aplicație a IMS MS la studiul speciilor de condroitin/dermatan sulfat exprimate in creierul uman; c) descoperirea de noi secvențe CS/DS in matricea extracelulara umana; d) detectarea și investigarea structurală a motivelor CS/DS sub- și suprasulfatate în diverși proteoglicani, inclusiv în cei exprimați în sistemul nervos central; e) identificarea de noi secvențe CS/DS biologic active pe baza interacțiunilor lor functionale. Rezultatele obtinute au demonstrat ca IMS MS este o tehnică versatilă si integrată, ce permite o analiza rapida, totală si ultra-sensibilă astfel incat este de asteptat ca protocoalele dezvoltate în cadrul CHON-DERM-IMS sa găsească o aplicabilitate largă pentru studii legate de alte tipuri de biomolecule, in special biomarkeri ai unor afectiuni severe. .