pngase f solution  (New England Biolabs)

Journal: bioRxiv

Article Title: Mass Spectrometry Imaging of the Subchondral Bone in Osteoarthritis Reveals Tissue Remodeling of Extracellular Matrix Proteins that Precede Cartilage Loss

doi: 10.1101/2024.08.03.606482

Figure Lengend Snippet: (A) Non-arthritic Cadaveric Joint (control) cadaveric tissue donors without diagnosed arthritis, osteoporosis, or fracture, and Osteoarthritis (OA) patients receiving knee replacement compose a cohort that is age, sex, and BMI matched and stratified by their clinical OARSI (arthritis) grade. Safranin-O/Fast Green histologic staining (B) of the tibial plateau of a non-arthritic donor (Control - top) and a representative patient with Osteoarthritis (OA - bottom) depict the loss of proteoglycan staining (red) in Medial OA, indicative of cartilage loss. Separation of tissue into medial and lateral sections allows for both site-matched comparisons with cadaveric tissue (medial OA vs Medial Control, blue arrow) and tissue-matched comparisons to healthier tissue from the same OA patients (OA Medial vs OA Lateral, red arrow). Formalin-fixed, paraffin-embedded (FFPE) tissue sections are prepared (C) for MALDI-MSI spatial proteomics including deparaffinization and epitope retrieval prior to PNGase F enzyme treatment for removal of proteoglycans. Tissues are matrix-coated and imaged for glycomics before being washed and prepared for peptide imaging following a similar protocol utilizing collagenase III that targets the skeletal ECM. Following matrix application, slides are imaged (D) with the timsTOF fleX mass spectrometer (Bruker).

Article Snippet: Deglycosylation was achieved by applying a 0.1 μg/μL PNGase F solution (N-Zyme Scientifics) via the TM Sprayer M3 (HTX Imaging, Chapel Hill, NC, USA), followed by incubation in custom humid chambers for 2 hours at 37°C.

Techniques: Control, Staining, Formalin-fixed Paraffin-Embedded, Imaging, Mass Spectrometry