Improved detection of botulinum type E by rational design of a new peptide substrate for endopeptidase–mass spectrometry assay

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

“…c o m / l o c a t e / y a b i o High sensitivity and a broad selectivity range have been reported for different BoNT serotypes tested in different media (buffer, food, serum, and stool) using the Endopep-MS assay [17][18][19][20]. Recently, we reported on the design of a new peptide substrate for improved detection of BoNT type E (BoNT/E) [21]. Our strategy was based on previously reported BoNT/E-SNAP-25 interactions integrated with efficiency considerations of the analysis method, that is, liquid chromatography-tandem mass spectrometry/multiple reaction monitoring (LC-MS-MS/MRM).…”

“…Our strategy was based on previously reported BoNT/E-SNAP-25 interactions integrated with efficiency considerations of the analysis method, that is, liquid chromatography-tandem mass spectrometry/multiple reaction monitoring (LC-MS-MS/MRM). Applying the new modified substrate resulted in significantly increased sensitivity (>1 order of magnitude) over assays using the earlier substrate [21]. Here we describe the design of a new peptide substrate for BoNT type B (BoNT/B) Endopep-MS assay that led to enhanced detection of the toxin.…”

Early, Real-Time Medical Diagnosis of Botulism by Endopeptidase-Mass Spectrometry

“…Beads were then thoroughly washed ( Figure 1B, step 2) and transferred to the endopeptidase reaction (20 µL) that contained 1 mM ZnCl 2 , 10 mM dithiothreitol, 1% Triton, 0.1 mM peptide substrate (K(BIO-AHX)GSNRTRIDEGNQRATR(Nle)LGG, LSELD-DRADALQAGASQFETSAAKLKRK, and (Nle)GNEIDTQNR-QIDRI(Nle)EKAD for A [9], B [7], and E [6], respectively) and 50 mM Hepes buffer ( pH 7.4) at 37°C for 5 hours ( Figure 1B, step 3). The reaction was terminated by the addition of 180 µL of 1% formic acid and heating to 95°C for 2 minutes.…”

“…The reaction was terminated by the addition of 180 µL of 1% formic acid and heating to 95°C for 2 minutes. The cleaved products were detected as previously described by LC-MS/MS (MRM) ( Figure 1B, step 4) [6,7].…”

Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

“…The peptides of Ile 156 -Asp 186 and Ile 156 -Thr 190 are used in a mass spectrometry-based Endopep-MS assay developed in our laboratory[23; 24]. During the preparation of this manuscript, a new paper published claimed the peptide of Met 167 -Asp 186 and its derivative with two Met replaced by Nle residues were effective substrates for the Endopep-MS platform[25]. This report described the development of a novel peptide substrate to improve the sensitivity of the Endopep-MS assay for the determination of BoNT/E catalytic activity.…”