Chymotrypsin digest and LC–MS/MS

Each in-solution sample was denatured in 150 µl of ice-cold lysis buffer (50 mM Tris-formic acid, 150 mM NaCl, 0.5% sodium deoxycholate, 2% SDS, 2% NP-40, pH 8.0). An acetone precipitation/on-pellet-digestion procedure was applied to perform precipitation and tryptic digestion for reproducible peptide recovery, as described previously^35,36.

The peptide mixture was separated and analyzed using an UltiMate 3000 RSLCnano-LC system coupled with an Orbitrap Fusion Lumos mass spectrometer. The mobile phase A contains 2% acetonitrile in 0.1% formic acid, and mobile phase B contains 88% acetonitrile in 0.1% formic acid. 4 µl of digested samples were loaded onto a large-ID trap (300 µm ID × 0.5 cm, packed with Zorbax 5-µm C18 particles) with 1% B at a flow rate of 10 µl/min for 3 min. The trapped peptides were then back-flushed onto the nano-LC column heated at 52 °C (75 µm ID × 60 cm, packed with Waters XSelect CSH 2.5-µm C18 particles) at a flow rate of 250 nl/min.

Mass spectrometry data were acquired under data-dependent and positive ion scan mode with a spray voltage of 2000 V, ion transfer tube temperature of 250 °C, and a cycle time of 3 s. One scan cycle included a survey scan (m/z 400–1500) at a resolution of 120,000 with an AGC target of 4 × 10⁵ and a maximum injection time of 50 ms. MS2 was performed by an isolation window of 1.2 Th with the quadrupole for high energy collision dissociation (HCD) fragmentation and detected by Orbitrap at a resolution of 15,000 with an AGC target of 5 × 10⁴. The maximum injection time was 50 ms, and the collision energy was 30%. Dynamic exclusion was enabled with a repeat count of 1 and an exclusion duration of 45 s.

The mass spectrometry raw files were searched against CYP121A1 protein sequence and its background of Escherichia coli database from UniProt using the Proteome Discoverer v1.4 (https://www.thermofisher.com/). The false discovery rate was determined by using a target-decoy search strategy. Scaffold 4.5 (Proteome Software, Portland, OR; http://www.proteomesoftware.com/) was used to validate MS/MS-based peptide and protein identification. The false discovery rates of 0.1% at the peptide level, a minimum of two unique peptides, and only 1 decoy protein were applied in this study. An ion current-based quantification method was used as previously described^37,38. Thresholds of 2.0-fold change and a p-value cutoff of 0.05 were used to define the altered peptides. To determine the crosslinking sites between the glutaraldehyde-treated and control group, a minimum of two unique peptides were required to support the alteration.