Fig. 8: Generation of hTTR(59–127) in human plasma. | Communications Biology

Fig. 8: Generation of hTTR(59–127) in human plasma.

From: A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment

Fig. 8

Aliquots (800 μl) of human plasma were treated with subtilisin at different concentrations (0, 0.18, and 0.72 μM) at 37 °C under constant agitation (500 rpm). After 16-h reaction, plasma samples were processed following the phenol precipitation protocol (see “Methods” section) and analysed by SDS-PAGE and UPLC-MS. a SDS-PAGE (4–14% acrylamide) analysis under reducing conditions of phenol-treated plasma samples (8 μg). Proteins were identified by in situ tryptic digestion and peptide mass fingerprint analysis (see “Methods” section): 1, IgG heavy chain; 2, α1-acid-glycoprotein 1; 3, IgG light chain; 4, hTTR; 5, hTTR(59–127). b Representative UPLC-MS chromatographic profiles of phenol-treated plasma samples before (A) and after (B, C) 16-h incubation with increasing subtilisin concentrations. In each panel, the black trace reports the total ion current (TIC) profile, expressed in percentage, while the blue and red traces track the intensities of the highest m/z values of S-Cys-hTTR (m/z = 817.472) and hTTR(59–127) (m/z = 970.492) in the eXtracted Ions Chromatograms (XIC). The arrows refer to the retention times in the XICs of phenol-treated plasma samples spiked with hTTR (blue arrow) or hTTR(59–127) (red arrow).

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