Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin.

USP's peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.

Onset of oxidative damage in alpha-crystallin by radical probe mass spectrometry.

The early onset oxidative damage within segments of the protein alpha-crystallin is examined by radical probe mass spectrometry by its treatment with reactive oxygen species under low-, moderate-, and high-oxidizing conditions. Five regions comprising the first 11 residues of the N-termini of the A and B subunits (A/B:1-11), central domains from each subunit B:57-69 and A:104-112, and a C-terminal segment of the A subunit A:120-145 were found to be the initial sites of oxidation. The susceptibility of each segment to oxidation and oxidative damage is investigated by subjecting the intact protein to different oxidation conditions within the ion source of an electrospray ionization mass spectrometer. LC-MS of the oxidized protein digests enables the sites and levels of oxidation to be monitored. The onset of oxidative damage and the levels of oxidation observed before damage occurs differ across the protein surface. The regions comprising residues A/B:1-11 and A:104-112 are shown to be more susceptible to oxidative damage than those comprising residues B:57-69 and A:120-145. The results are discussed in the context of available experimental and homology-modeled theoretical structures for the subunits of alpha-crystallin.