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

Ching-Hsin Tung ^a,b, Chih-Chieh Lin ^a, Ching-Chuan Tung ^c, Sung-Fang Chen ^d, Fuu Sheu ^e, Ting-Jang Lu ^a,*

^a Graduate Institute of Food Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan, ROC

^b Food and Drug Administration, Ministry of Health and Welfare, No.161-2, Kunyang St, Nangang District, Taipei 11561, Taiwan, ROC

^c Department of Nursing, Chang Gung University of Science and Technology, No. 261, Wenhua 1st Road, Guishan, Taoyuan, 33303, Taiwan, ROC

^d Department of Chemistry, National Taiwan Normal University, No. 88, Sec. 4, Ting-Chow Road, Taipei, 11677 Taiwan, ROC

^e Department of Horticulture, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan, ROC

A rapid analytical approach, on-line desalting HPLC-UV-ESI-MS method, for the analysis of FIP-fve and flammutoxin (FTX), two important bioactive proteins in the fruiting bodies of Flammulina velutipes, was developed. In this study, a highly efficient desalting method is provided using molecular weight cut-off centrifugal filtration and on-line desalting. Sample preparation followed by an on-line desalting HPLC-UV-ESI-MS system was employed for simultaneous desalting and detection and identification of FIP-fve and FTX. Results indicated that using trifluoroacetic acid as a modifier on a C18 reversed-phase column renders effective separation. ESI-MS revealed that the apparent molecular masses of FIP-fve and FTX were 12,749.1 Da and 21,912.5 Da, respectively. Eleven milligrams of FIP-fve was obtained from 100 g of fresh fruiting bodies, and UV detection was performed at 280 nm using bovine serum albumin as the standard protein. The calibration curve was linear in the concentration range of 0.29-4.69 mg/mL (r2 ¼ 0.9999). FTX and a series of degradation products were isolated from F. velutipes using 35% saturated ammonium sulfate on a DEAE cellulose column. The complete identification of FTX and a series of degradation products were carried out by precipitation of various ammonium sulfate concentrations (0-45%, 45-65% and 65-90%), in-gel trypsin digestion, and MS analysis with combined database search. The molecular weights of FTX and a series of degradation products were 29,957.2 Da, 27,480.2 Da, 26,512.5 Da, and 21,912.5 Da.

Keywords: FIP-fve, Flammulina velutipes, Flammutoxin, Fruiting bodies