One nanogram of BoNT/FA was added to peptide substrate LSELDDRADALQAGASQFESSAAKLKRKYWWKNLK (SubB) in the presence of reaction buffer as described previously (24 (link)). Another aliquot of 1 ng of BoNT/FA was added to another peptide substrate TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL (SubF) also in the presence of reaction buffer. Following a 4-h incubation at 37°C, a 2-µl aliquot of each reaction supernatant was mixed with 18 µl of matrix solution, and a 0.5-µl aliquot of this mixture was pipetted onto one spot of a MALDI plate as described previously (24 (link)). Mass spectra of each spot were obtained by scanning from 900 to 5500 m/z in MS-positive ion reflector mode on an Applied Biosystems 5800 proteomics analyzer (Framingham, MA). The instrument uses an Nd-YAG laser at 355 nm, and each spectrum is an average of 2,400 laser shots.
To positively identify BoNT/FA via its amino acid sequence, 1 µg of the toxin was added to 13 µl of 100 mM ammonium bicarbonate at pH 7.5 and 2 µl of trypsin at 0.5 mg/ml in water. Following mixing, the mixture was heated at 52°C for 10 min, followed by the addition of 1 µl of 10% of trifluoroacetic acid. Peptides from the toxin were identified by LC-MS/MS with database searching as described previously (20 (link)).
To positively identify BoNT/FA via its amino acid sequence, 1 µg of the toxin was added to 13 µl of 100 mM ammonium bicarbonate at pH 7.5 and 2 µl of trypsin at 0.5 mg/ml in water. Following mixing, the mixture was heated at 52°C for 10 min, followed by the addition of 1 µl of 10% of trifluoroacetic acid. Peptides from the toxin were identified by LC-MS/MS with database searching as described previously (20 (link)).
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