HPLC analysis and quantification

An Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) controlled using ChemStation software and equipped with a PDA detector was used. Incubations were monitored at 280 and 216 nm. Separation of the peptides was achieved using a Jupiter Proteo 4 μm 90 Å column (150 mm × 4.6 mm ID) at 40°C. The gradient was 0% B (0.1% TFA in acetonitrile) to 60% B (0.1% TFA in acetonitrile) over 15 min with a flow rate of 2 mL min⁻¹. The degree of modification was calculated based on the moles of each peptide formed as a percentage of moles of peptide present at the start of the incubation. The error bars refer to the standard deviation of three replicates. Separation of peptides in the alpha B crystallin incubations was achieved using the same gradient but run over two hours.