Orbitrap fusion lumos tribrid

Cross-linked peptide samples resuspended in 5% DMSO/10% FA/85% H₂O were separated by a nanoflow LC system (Dionex UltiMate 3000 RSLC nano), utilizing the same gradient as above. The nano column (EASY-Spray PepMap RSLC C18, 2 μm 100 Å, 75 μm × 50 cm), set at 40 °C, was connected to an EASY-Spray ion source (Thermo Scientific). Spectra were collected from an Orbitrap mass analyzer (Orbitrap Fusion Lumos Tribrid, Thermo Scientific) using full MS mode (resolution of 60,000 at 400 m/z) over the mass-to-charge (m/z) range 375 to 1600, utilizing an XLMS cleavable MS2-MS3 method in the case of DSSO, or an MS2 only method for DMTMM. Peptides of charge 3 to 8 were chosen and sorted, favoring the highest charged state. Data-dependent MS2 scan was performed using Quadrupole isolation with a cycle time of 5 s with CID activation and Orbitrap detection at 30,000. A targeted mass difference of Delta M1: 31.9721 was defined to detect the presence of DSSO cross-linked peptides for their analysis in an MS3 scan. Data-dependent MS3 scans were performed using Quadrupole isolation with CID activation and ion trap detection.

The fractions were analyzed using an Orbitrap Fusion Lumos Tribrid mass spectrometer (MS) interfaced with an Easy-nLC1200 liquid chromatography system (Thermo Fisher Scientific). Peptides were trapped on an Acclaim Pepmap 100 C₁₈ trap column (100 μm by 2 cm; particle size, 5 μm; Thermo Fisher Scientific) and separated on an in-house packed analytical column (75 μm by 35 cm; particle size, 3 μm; Reprosil-Pur C₁₈ [Dr. Maisch]), using a linear gradient from 5% to 33% solvent B over 77 min followed by an increase to 100% solvent B for 3 min and then 100% solvent B for 10 min at a flow rate of 300 nL/min. Solvent A was 0.2% formic acid, and solvent B was 80% acetonitrile, 0.2% formic acid. Precursor ion mass spectra were acquired at 120,000× resolution, and MS/MS analysis was performed in a data-dependent multinotch mode, wherein collision-induced dissociation (CID) spectra of the most intense precursor ions were recorded in ion trap at a collision energy setting of 35 for 3 s (top speed setting). Precursors were isolated in the quadrupole with a 0.7 m/z isolation window, charge states 2 to 7 were selected for fragmentation, and dynamic exclusion was set to 45 s and 10 ppm. MS3 spectra for reporter ion quantitation were recorded at 50,000× resolution with high-energy collisional dissociation (HCD) fragmentation at a collision energy of 65, using the synchronous precursor selection.