Magic aq c18 material

MRM assays [89 (link)] were designed to validate the differentially expressed protein, CAPG in RA and OA synovial fluid. Skyline version 2.1 [90 (link)] was used for method development and optimization. The target peptide selected for CAPG was QAALQVAEGFISR (z = +2, m/z = 695.38) and top four transitions monitored included, y8⁺ → 878.47, y7⁺ → 779.40, y6⁺ → 708.37 and y5⁺ → 579.32. The samples were subjected to in-solution digestion as described earlier [91 (link)]. All samples were analyzed in triplicate on TSQ Quantum Ultra (Thermo Scientific, San Jose, CA) interfaced with Easy nanoLC II (previously Proxeon, Thermo Scientific, Bremen, Germany). The peptides were enriched on a trap column (5 μm, 75 μm × 2 cm.) with 0.1% formic acid and 5% ACN for 5 minutes and separated on an analytical column (3 μm, 75 μm × 10 cm) with an increasing linear gradient from 5-35% of solvent B (90% ACN in 0.1% formic acid) for 60 min at a constant flow rate of 300 nl/min. Both columns were packed in-house using Magic AQ C18 material (Michrom Bioresources). Spray voltage of 2.5 kV was applied and ion transfer tube was maintained at 275°C. The data was acquired with Q1 and Q3 set at resolution of 0.4 and 0.7 respectively. The collision energy for each transition was optimized with the help of Skyline based on the preliminary results [90 (link)].