LC–MS3 analysis of DSSO cross-linked peptides

The SEC–HpHt fractions were subjected to LC–MS³ analysis using an UltiMate 3000 RSLC nano-HPLC system coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific), as described previously²³. Peptides were separated by reversed-phase LC (50 cm × 75 μm Acclaim PepMap C18 column, Thermo Scientific) with over an 87-min gradient of ACN (4% to 25%) at a flow rate of 300 nl min^–1. Initial survey (MS¹) scans were measured in the Orbitrap with a scan range from 375 to 1,800 m/z, a resolution of 60,000 FWHM and an AGC target of 4 × 10⁵ with a maximum injection time of 75 ms at top speed per 4 s of cycle time. Ions with a charge of 4⁺ or greater were selected for MS² and subjected to fragmentation using collision-induced dissociation with a normalized collision energy of 23%. For MS² scans, the scan range was set to auto mode, with a resolution of 30,000 FWHM, an AGC target of 5 × 10⁴, a precursor isolation width of 1.6 m/z and a maximum injection time of 100 ms. A targeted inclusion on ions with a mass difference corresponding to the difference in alkene and thiol DSSO fragments (31.9721 Da) was used to select precursors for MS³ analysis. For MS³ scans, higher collision-induced dissociation was used with a normalized collision energy of 28%, the AGC target was set to 2 × 10⁴, and the maximum injection time was set to 125 ms.