Mass Spectrometry Protocol for Protein Identification

MS analysis was performed as described by Zhang et al. [41 (link)] and Zheng et al. [42 (link)] with some modifications. Each protein solution was mixed with an equal volume of matrix solution (70% acetonitrile, 0.1% trifluoroacetic acid, 10 mg/mL α-cyano-4 hydroxycinnamic acid). Mass spectra were analyzed using MALDI-TOF mass spectrometer (BrukerDaltonics, Bremen, Germany). Sequence query using peptide mass values and corresponding fragment peak lists were used to search for protein sequences against the NCBInr and Swissport databases using MASCOT version 2.3 software (Matrix Science, Franklin, UK) with the following search parameters: taxonomy, all series, allowed modifications, carbamidomethyl of cysteine (fixed), oxidation of methionine (variable), peptide tolerance, ±0.3 Da. The highest MOWSE score was only considered as the most probable identification, and was significant (p < 0.05) when protein scores were >88 (NCBInr) or 70 (Swissport).