More oxidation was caused by freeze–thawing 10 times over 14 d at −20 °C (Fig. 5d), or leaving the peptide at −20 °C

over 80 d (Fig. 5e), or leaving the peptide at 4 °C for 37 months (Fig. 5f). Storing peptide III_24 in N2-saturated solution with repeated freeze–thawing over 14 d slowed oxidation four-fold (data not shown). Long-term storage of other Toolkit peptides resulted in variable polymerization. Just 12% of 37 month-old III-04 had formed helical polymers while 44% of 41-month old II-56 was polymeric, similar to the level shown for III-24 in Fig. 5f. A sample of III-24 (Tm 51 °C at 2.5 mg mL−1) stored for 48 months at 4 °C was 47% triple-helical when analyzed by gel filtration at 40 °C. However, after 10 min reduction with 2 mM TCEP, the proportion of triple-helical peptide was 18%. Helicity was ∼75% if gel filtration was run at 10 °C, regardless of the presence of TCEP. Peptides were heat-denatured after storage Fasudil at 4 °C for 9 months or longer. They were analyzed by gel filtration at 60 °C, and by MALDI and electrospray mass spectrometry immediately after heating to 60 °C. Their cysteine thiol content was determined using Ellman’s reagent. This allowed Compound C in vitro us to characterize the peptide polymer mixture (Suppl. Figs. S2–S5, Tables S1 and S2, Sections 3.8, 4.4, 4.5). Briefly, >90% of

cysteine in peptides aged for 9 months or more is oxidized, and cross-linked such that 5–13% of the peptide is monomeric (mostly cyclic), 7–50% is dimeric, correlating with peptide stability and purity, where CRPcys has less dimer than the other peptides, and the remainder is polymeric. Positive controls using

fresh peptide were ∼95% reduced as expected. Gel filtration revealed that, in the presence of 2 mM TCEP, peptide III-24 at 2.5 mg mL−1 Myosin was almost free of any component bigger than a single helix, no matter what temperature (4–50 °C) was maintained before loading onto the column (see, for example, Fig. 5a). To confirm this, we undertook DLS experiments under reducing conditions in neutral buffer. There was no evidence of any species larger than around 16.5 kDa, equivalent to a single helix. We could not resolve peptide monomer from helix, so mass and Stokes Radius shown in Table 2 represent average values, decreasing with increasing temperature due to helix denaturation. Stokes Radius correlated well with values obtained from gel filtration, and are as expected for rod-like molecules of this mass. We evaluated the coating of biotinylated peptides with or without cysteine to 96-well plates, detected as described in Section 2. We could detect coating of the plastic by cysteine-containing biotinylated peptide (B-GFOGERcys), but biotinylated peptides lacking cysteine-adhered poorly (B-GFOGER) or not at all (B-CRP) (Fig. 6a). Additionally, all peptides containing motifs that bind integrin α2β1 or GpVI and terminal cysteine supported platelet adhesion (CRPcys, GFOGERcys, B-GFOGERcys, Fig. 6b).