Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue

Abstract

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.

Complex-Type N-Glycans Are Associated with Cartilage Degeneration within Different Loading Sites of the Tibial Plateau for Knee Osteoarthritis Patients

“…Consecutive FFPE osteochondral tissue sections from both the lateral and medial compartments of the tibial plateau were used for MALDI-MSI analysis. Each section was mounted onto a gelatin-coated indium tin oxide (ITO) slide. , Sample preparation entailed deparaffinization, rehydration, overnight gentle antigen retrieval, PNGase F enzyme application, incubation, and α-cyano-4-hydroxycinnamic acid (HCCA) matrix deposition (see Table S2 for spray-specific settings). A detailed protocol is available in our recently published papers. , …”

“…Each section was mounted onto a gelatin-coated indium tin oxide (ITO) slide. , Sample preparation entailed deparaffinization, rehydration, overnight gentle antigen retrieval, PNGase F enzyme application, incubation, and α-cyano-4-hydroxycinnamic acid (HCCA) matrix deposition (see Table S2 for spray-specific settings). A detailed protocol is available in our recently published papers. , …”

“…Ion intensity maps were weakly denoised with automatic hotspot removal. Annotation of putative N -glycan structures was performed as previously described . Only N -glycans found in the GlyConnect database were selected and then created using Glycoworkbench (v2.1) …”

“…detailed protocol is available in our recently published papers. 10,15 Data were acquired using a timsTOF fleX MS instrument (Bruker Daltonics, Bremen, Germany) controlled by timsControl (v3.0, Bruker Daltonics, Bremen, Germany) and flexImaging (v7.0, Bruker Daltonics, Bremen, Germany) in positive reflectron mode. Laser power was set at 60%, and spectra were acquired at 500 shots per pixel with a pixel size of 50 μm across a m/z range between 700 and 3200.…”

“…We recently reported, for the first time, that complex-type N -glycans, (Hex) 4 (HexNAc) 3 , (Hex) 4 (HexNAc) 4 , and (Hex) 5 (HexNAc) 4 , were increased in formalin-fixed paraffin-embedded (FFPE) human KOA cartilage tissue ( n = 3) compared to cadaveric controls ( n = 3) using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) . Interestingly, these N -glycans were predominantly localized in the superficial fibrillated zone of the degenerate cartilage.…”