Peptide Stability Evaluation in Human Plasma

Peptide stability tests were performed according to a TCA stability assay based on the method used by Nguyen et al. [38 (link)]. Peptides were dissolved in water, mixed with human plasma* to a final concentration of 1.0 mg/mL, and incubated at 37 °C with agitation (300 rpm, Thermomixer, Eppendorf AG, Hamburg, Germany). After 0, 1, 2, 3, 6 and 24 h, 80 μL of the solution was taken and mixed with 15% TCA to obtain a final concentration of 3% (v/v). The samples were incubated in ice for 10 min and centrifuged for 10 min (12,000 rpm, Microfuge 16, Beckman Coulter, Brea, CA, USA). Supernatants were analyzed by RP-HPLC on column using a linear gradient of acetonitrile (5–100% over 15 min) with detection at 223 nm. Peptides were quantified by their peak areas relative to the initial peak areas (0 min). All stability tests were performed at least in triplicates. As control samples peptides dissolved only in water under the same conditions were used. To additionally assess the changes in the incubated samples (peptide degradation) mass spectrometry analysis was used. LC-MS experiments were performed using ESI-IT-TOF (Shimadzu, Kyoto, Japan).
* Blood was collected from healthy anonymous donors and EDTA was then used as an anticoagulant. The procedure was approved by the Independent Bioethics Commission for Research of the Medical University of Gdańsk (NKBBN/387/2014).