2.4. Liquid Chromatography Tandem Mass Spectrum (LC-MS/MS) Analysis of psRFP

The purified protein (75 μM) was reacted with 300 μM HS_nH or DTT at 25 °C for 1 h. The reacted mixture was loaded onto a PD-10 desalting column to remove unreacted small molecules. The re-purified proteins (≤100 μg) were then mixed with 50 μL denaturing buffer (0.5 M Tris-HCl, 2.75 mM Ethylenediaminetetraacetic acid (EDTA), 6 M Guanadine-HCl, pH 8.1) and 50 μL 1 M iodoacetamide (IAM) in a dark place for 1 h. 360 μL of 25 mM NH₄HCO₃ was used to wash the mixture four times in a Microcon YM-10 K centrifugal filter unit (Millipore, Billerica, MA, USA). The washed proteins were then digested with trypsin by following a previously reported protocol [22].

The Prominence nano-LC system (Shimadzu, Kyoto, Japan) equipped with a custom-made silica column (75 μm × 15 cm) packed with 3 μm Reprosil-Pur 120 C18-AQ was used for the analysis. For the elution process, a 100 min gradient from 0% to 100% of solvent B (0.1% formic acid in 98% acetonitrile) at 300 nL/min was used, and solvent A was 0.1% formic acid in 2% acetonitrile. The eluent was ionized and electro-sprayed via LTQ-Orbitrap Velos Pro CID mass spectrometer (Thermo Scientific, Waltham, MA, USA), which run in data-dependent acquisition mode with Xcalibur 2.2.0 software (Thermo Scientific, Waltham, MA, USA). The spray voltage was of −3.0 and 3.0 kV in negative and positive modes, respectively. The ion transfer tube temperature was set at 300 °C. Full-scan mass spectra (from 400 to 1800 m/z) were detected in the Orbitrap with a resolution of 60,000 at 400 m/z.