Combination of on-line desalting and HPLC-UVESI- MS for simultaneous detection and identification of FIP-fve and flammutoxin in Flammulina velutipes

A rapid analytical approach, on-line desalting HPLC-UV-ESI-MS method, for the analysis ofFIP-fve and flammutoxin (FTX), two important bioactive proteins in the fruiting bodies ofFlammulina velutipes, was developed. In this study, a highly efficient desalting method isprovided using molecular weight cut-off centrifugal filtration and on-line desalting. Samplepreparation followed by an on-line desalting HPLC-UV-ESI-MS system was employed forsimultaneous desalting and detection and identification of FIP-fve and FTX. Resultsindicated that using trifluoroacetic acid as a modifier on a C18 reversed-phase columnrenders effective separation. ESI-MS revealed that the apparent molecular masses ofFIP-fve and FTX were 12,749.1 Da and 21,912.5 Da, respectively. Eleven milligrams of FIP-fvewas obtained from 100 g of fresh fruiting bodies, and UV detection was performed at280 nm using bovine serum albumin as the standard protein. The calibration curve waslinear in the concentration range of 0.29e4.69 mg/mL (r2 = 0.9999). FTX and a series ofdegradation products were isolated from F. velutipes using 35% saturated ammoniumsulfate on a DEAE cellulose column. The complete identification of FTX and a series ofdegradation products were carried out by precipitation of various ammonium sulfateconcentrations (0e45%, 45e65% and 65e90%), in-gel trypsin digestion, and MS analysiswith combined database search. The molecular weights of FTX and a series of degradationproducts were 29,957.2 Da, 27,480.2 Da, 26,512.5 Da, and 21,912.5 Da.