Half-life determinations require an estimate of deamidation rate constants based on the local 2D and 3D protein structure. Protein crystal structures were available through the Protein Data Bank (www.rcsb.org/pdb) for portions of some proteins (FN1 and COMP) or generated using the I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER) (24, 56). Swiss-PdbViewer (v4.10) (57) was used to visualize the structures and manually acquire the steric covariate values for estimating deamidation rate (16, 34). Deamidation, like racemization, is a first-order process (17). Therefore, accumulation of deamidated residues can be described by two linear processes: the rate constants of deamidation (Kid) and the protein turnover (KT). The algorithm can be described asdCAsn(D/D+N))/dt=KidCAsnN/(D+N)-KTCAsnN/D/(D+N)where CAsn is the molar concentration of Asn in the protein, and D/(D + N) and N/(D + N) represent the fractions of the deamidated and nondeamidated native proteins. The measured amount of deamidated residues in the sample is the net result of accumulated deamidated residue accumulation and their loss due to protein turnover. The original estimation of deamidation rates was reported in days (17). Therefore, the deamidation rates derived from our algorithm also roughly correspond to units of days.

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