Mass spectrometry (MS) analysis

The single bands of protein subunits were obtained by SDS-PAGE electrophoresis and cut out, subjected to mass analysis using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer MALDI-TOF/TOF (UltraflextremeTM, Brucker, Germany). The protein samples were first treated with DTT reduction and alkylation with iodoacetamide, followed by treatment with trypsin overnight⁵¹. The peptides obtained after enzymatic hydrolysis were desalted by a C18 ZipTip and mixed with the matrix-cyano-4-hydroxycinnamic acid, followed by mass analysis. Database searches were performed through the MS/MS Ion Search page at www.matrixscience.com. The protein identification results were obtained based on the primary mass spectrometry and the secondary mass spectra of the peptides produced after enzymatic hydrolysis. The database used for the identification is NCBIProt, swissprot.