A modified peptide mapping strategy for quantifying site-specific deamidation by electrospray time-of-flight mass spectrometry

A modified peptide mapping strategy using electrospray time‐of‐flight mass spectrometry with high‐performance liquid chromatography (HPLC/MS) provides an improved measure of deamidation by performing proteolytic digestion at low temperature (4°C), low pH (6.0) and in organic solvent (≥10% acetonitrile). HPLC resolution of the native (N) and deamidated (D) peptides is achieved, and the ratio of ion counts is converted into percent deamidation. The percent deamidation is established for a reference lot using a time course of digestion (24–120 h) and extrapolation to time zero. Test samples are compared against the reference lot to quantitate changes in site‐specific deamidation. A recombinant purified protein (antigen A) having a single labile Asn‐Gly site is analyzed using this strategy. The N and D peptides from an endoproteinase Lys C (Lys C) digestion (pH 6, 4°C) resolve to near homogeneity on HPLC which results in equivalent percent deamidation when calculated by either UV or ion counts. Deamidation increases with time and pH of proteolysis. Lys C peptide maps of antigen A and bovine serum albumin (BSA) digested at pH 5–8 are comparable. A Lys C digestion time course of a reference lot of antigen A extrapolates to 18% deamidation of the Asn‐Gly site at time zero. This strategy may be generally applicable to protease–protein combinations for improved accuracy in measuring site‐specific deamidation by peptide mapping LC/MS. Copyright © 2007 John Wiley & Sons, Ltd.