Automated evaluation of tryptic digest from recombinant human growth hormone using ultraviolet spectra and numeric...

UV spectra were successfully employed in identifying peptide fragments from a tryptic digest of recombinant-DNA-derived human growth hormone (r-hGH). It was possible to distinguish very similar peptides utilizing a digital comparison of the UV spectra. An automated procedure was developed to generate a calibration library for the tryptic digest of a reference standard. The calibration library was then evaluated for reproducibility and selectivity and found to provide superior performance in correctly identifying ambiguous peaks as compared to the use of a conventional calibration table. Spectral match factors together with numerical information, derived from peak retention time, area and height, were used to arrive at a "peak score" descriptive of the similarity between standard and sample peaks. "Peak scores" could be combined to calculate a "sample score" indicative of overall similarity between an unknown and a standard. The scoring procedure was automated to generate a final report without operator intervention and successfully assigned appropriate scores to similar as well as dissimilar samples, e.g., native and oxidized r-hGH.