Micro bio spin 6 size exclusion spin column

The fluorophore (Atto‐488, Sigma) was added to the protein mixture at a final concentration of 5 mM after reducing the protein by adding 1 equivalent of TCEP, incubating for 30 min at 4°C, and removing the reducing agent by a desalting column (Bio‐Rad Laboratories, Ltd.). The labeling reaction was incubated for 2 h at 4°C while rotating.
The labeling process for all variants was always finalized with buffer exchange twice using a Micro Bio‐Spin 6 Size Exclusion Spin Column (Bio‐Rad Laboratories, Ltd.) to remove the free spin label. The second buffer exchange was with 20 mM Tris–HCl (pH = 7.5), D₂O‐based, for pulse‐EPR measurements, unless stated otherwise.

For protein identification, protein bands were excised from a 10% Tricine-SDS-PAGE gel and subjected to in-gel tryptic digestion followed by peptide identification in a MALDI-TOF/TOF (matrix-assisted laser desorption/ionization-time of flight) analyzer (Applied Biosystems 4800plus). The data were analyzed in a combined mode using Mascot search engine and National Center for Biotechnology Information (NCBI) database.
For intact mass determination, DsrD was analyzed before and after addition to the sulfite reductase assay. The protein buffer was exchanged to 20 mM ammonium acetate (pH 7.2) before analysis, using a Micro Bio-Spin 6 size exclusion spin column (Bio-Rad). The protein solution was mixed with 10 mg/mL Sinapinic acid (Sigma) in 50% (vol/vol) acetonitrile and 5% (vol/vol) formic acid (liquid chromatography–mass spectrometry (LC/MS) grade; Fisher) in a 1:1 ratio, and then this solution was applied directly onto the MALDI plate and allowed to air dry. The data were acquired in Linear Mid Mass Positive mode using a 5800 MALDI-TOF/TOF (AB Sciex) mass spectrometer and TOF/TOF Series Explorer Software v.4.1.0 (AB Sciex). External calibration was performed using a Protein MALDI-MS Calibration Kit (MSCAL3, ProteoMass). MS data were obtained by the ITQB/iBET UniMS Mass Spectrometry Unit, Oeiras, Portugal.