Male ab plasma

We incubated the control and Ca-ion samples in a 24-well NUNC plates (Thermo Fisher Scientific, Waltham, MA, USA) for 3 h (37 °C, 5% CO₂) with 1 mL of human blood serum from male AB plasma (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany). In order to allow a standard, replicable characterization, we used commercial human serum as previously described in Romero-Gavilán et al. [22 (link)]. After 3 h incubation, we removed the serum and washed the discs five times with ddH₂O and once with 100 mM NaCl, 50 mM Tris–HCl (pH 7.0) to eliminate non-adsorbed proteins. We collected the adsorbed protein layer by washing the discs with an elution (0.5 M triethylammonium bicarbonate buffer (TEAB), 4% of sodium dodecyl sulfate, 100 mM of dithiothreitol (DTT)). We carried out four independent experiments for each type of surface, and we used four discs of each surface type in each experiment. We used a Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) to quantify the serum protein content, which was 50 μg/μL.

All peptides were tested on human serum from male AB plasma (Sigma–Aldrich) at an initial concentration of 300 μM. Peptide incubation time points were 0, 2, 4, 8, 16 and 24 h. Samples were prepared according to previously described methods [29 (link)]. Briefly, 90 μL of supernatant was taken at each time point and chromatographed on a 0.3 mL/min Phenomenex column using a linear 1%/min gradient of 0–50% solvent B on an analytical reverse-phase HPLC (Agilent). The elution times for all peptides were determined by the serum control at 0 h. The stability of each peptide for each time point was calculated as the height of the serum-treated peptide peak at 215 nm as percentage of the height of the 0-h serum-treated control. Experiments were done in triplicate.

The stability of peptides in human serum was examined using a protocol reported previously (Chan et al., 2011 (link)). Briefly, stock solutions of peptides (300 μM) were diluted 10 times with pre-warmed 100% human serum isolated from male AB plasma (Sigma-Aldrich) and incubated at 37°C for 0, 1, 2, 3, 5, 8, 12 and 24 h. Controls with peptides in PBS were included. The reaction was stopped by denaturing the serum proteins with urea at a final concentration of 3 M at 4°C for 10 min, followed by precipitation of serum proteins with trichloroacetic acid at a final concentration of 7% (v/v) (4°C, 10 min) and centrifugation (17,000 g, 10 min). The supernatant of each sample was recovered and run on an analytical column using a linear gradient of 5–30% solvent B (acetonitrile 90% (v/v) with 0.045% (v/v) TFA in H₂O) in solvent A (0.05% (v/v) TFA in H₂O) over 25 min at a flow rate of 0.3 mL/min with monitoring at 215 nm. The elution profile of each peptide was identified by the PBS sample from 0 time point. The percentage of peptide remaining in serum-treated samples was determined by comparing the height of the peptide peak obtained at each time point with that of the peptide peak obtained at 0 time point. Each experiment was done in triplicate.

The stability of
peptide ligands was
determined with human serum from male AB plasma (Sigma) centrifuged
at 13,300g for 10 min to remove the lipid content.
The supernatant was preincubated at 37 °C for 5 min before 10
μL of a peptide stock solution (3 mg/mL in H₂O) was
added to 300 μL of serum. A reference solution was prepared
in phosphate-buffered saline (PBS) buffer. The serum was incubated
at 37 °C and analyzed over a time course of 5 h with 40 μL
of aliquots removed at time points T = 0, 15, 30,
60, 90, 120, 240, and 300 min. Each aliquot was quenched with 40 μL
of 5 M urea and incubated on ice for 10 min. The proteins were then
precipitated with the addition of 40 μL of 20% (w/v) trichloroacetic
acid and further incubated on ice for 10 min. The samples were centrifuged
for 10 min at 15,000g, and the supernatant was analyzed
by analytical RP-HPLC using a Phenomenex Jupiter C₁₈ column
(5 μm, 300 Å, 150 × 2 mm) using a linear gradient
from 0 to 50% solvent B (90% acetonitrile, 10% H₂O, and
0.05% trifluoroacetic acid) and a flow rate of 1 mL/min. The percentage
of peptide remaining was determined by the integration of the analyte
in comparison to T = 0 min. The peptides were analyzed
in triplicate, and data were fitted to an exponential decay curve
using GraphPad Prism software.