Accepted answer
Because temperature enters the rate constant through an exponential, so equal steps in temperature multiply the rate instead of adding to it. Arrhenius puts the rate proportional to exp(−Ea/RT); the working approximation is a doubling per 10 K, which takes 5, 15, 25 and 35 °C to multipliers of 1, 2, 4 and 8. The steps in temperature are equal and the steps in rate are not, and that is the whole of the observation. At minus 20 °C the same rule gives no useful multiplier at all, because below freezing the reaction is no longer happening in bulk solution. A cyclic imide at Asp, eighteen daltons lighter, which then reopens to a mixture including the iso-aspartyl form — same formula as the parent, different molecule, and invisible to a mass-only method. Ea differs by route, so the ranking of routes changes with temperature too — which is why accelerated data extrapolates badly and why nobody should read a 40 °C study as a fast version of a 5 °C one.
The relevant point is that a mass shift of plus one dalton is deamidation and plus sixteen is oxidation, so degradation is often visible in a mass spectrum if anyone looks.
Freeze-thaw cycling drives aggregation through concentration at the ice interface and pH shifts as buffer components crystallise out at different rates. Each cycle costs something.
Aggregation is physical: peptides unfold at air-liquid interfaces and associate. Shaking maximises that interface, which is why swirling and shaking produce visibly different outcomes on the same vial.
Metal-catalysed oxidation of methionine is documented across peptide and protein formulations and is why chelators appear in some formulations.
Nothing here is medical advice, and research-use compounds are not approved for human use.
Swirl, never shake. Aggregation is a handling problem more than a time problem.
edited 21 Jan 2026 by sinead_gaffney — fixed an arithmetic slip in the third paragraph
4This should be linked from the help pages. – tri_gly_ala 4 months ago 3Confirming that opening a cold vial in a humid room is a genuinely bad idea. – mz_4113 2 months ago add a comment