Mz symmetry c18 trap column

MSC secretomes from all 12 donors for all treatment conditions (healthy, degenerative, traumatic, baseline, and IL-1β) were analyzed. The samples were collected and measured in two batches of 48 samples (traumatic, degenerative, IL-1β, baseline) and 24 samples (healthy and baseline). In both batches, baseline samples were included to account for differences between batches. For each sample, the protein concentration was measured using the Qubit® Protein Assay Kit (Life Technologies, Switzerland). The samples were then prepared by using a commercial iST Phonix Kit (PreOmics, Germany). Mass spectrometry analysis was performed on a Q-Exactive HF-X mass spectrometer (Thermo Scientific) equipped with a Digital PicoView source (New Objective) and coupled to an M-Class UPLC (Waters). Solvent composition at the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. For each sample, 4 μL of peptides were loaded on a commercial MZ Symmetry C18 Trap Column (100 Å, 5 μm, 180 μm × 20 mm, Waters) followed by nanoEase MZ C18 HSS T3 Column (100 Å, 1.8 μm, 75 μm × 250 mm, Waters). The peptides were eluted at a flow rate of 300 nL/min by a gradient from 8 to 27% B in 82 min, 35% B in 5 min, and 80% B in 1 min. Samples were acquired in a randomized order. Only precursors with intensity above 110,000 were selected for MS/MS.

For peptide separation by reverse phase chromatography, a Waters ACQUITY UPLC M-Class was used with a 20 mm nanoEase M/Z Symmetry C18 trap column (180 µm inner diameter packed with 5 µm C₁₈ silica particles), and a 25 cm nanoEase M/Z HSS C18 T3 column (75 µm inner diameter and packed with 1.8 µm C₁₈ silica particles), and analytical column. Total separation time was 75 min over a linear gradient from 5 to 35% solvent B (solvent A: 0.1% formic acid in H₂O, solvent B: 0.1% formic acid in AcN) with a flow rate of 300 nL/min. Separated peptides were directly applied by ESI into an Orbitrap Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). The mass spectrometry was set up in full MS/data-dependent-MS² mode. For the full scans, a scan range of 350–1500 m/z at a resolution of 120,000 was used with an automatic gain control (AGC) target at 3 × 10e6 and a maximum injection time of 50 ms. The top 20 most intense ions were isolated with a maximum injection time of 119 ms, fragmented with a NCE of 28 and detected at 60,000 resolution with a scan range of 200–2000 m/z and an AGC target of 1 × 10e5 (fixed first mass 130 m/z).