| Cysteine (important to protein stability) oxidizes to cystine in an oxidative environment, decreasing total cysteine in feed media within six days. What causes this loss in cysteine? To address this problem, a derivatizing reagent capable of detecting free cysteine, known as AccQ-Tag, was qualified with the Waters UPLC system. This method has repeatability and intermediate precision ≤5%, linear range from 31.3--625 microM, limit of quantitation between 15.6--31 microM, and spike recovery between 93-100%. Using this method, it was found that cysteine is rapidly converted to cystine and complete cysteine loss was determined in both basal and feed media. In the absence of cells and after six days, 50% of total cysteine and cystine is lost after feed media preparation. The loss of cysteine was due to cysteine to cystine conversion. This causes an increase in cystine and due to low cystine solubility, cystine became insoluble, leading to a loss of total cysteine. |
