Abstract
Liquid chromatography- tandem mass spectrometry (LC-MS/MS) is an important tool in characterizing the amino acid sequence and post-translational modifications of therapeutic antibodies. Identifying changes in the antibody is important in monitoring possible changes in activity and/or immunogenicity. However, sample preparation of antibodies for LC-MS/MS analysis can be difficult due to the tendency to form large aggregates, resistance to denaturing, and the formation of both very large and small peptides during enzymatic digestion. In this study, we have systematically investigated different trypsin digestion methods for chimeric immunoglobulin G (IgG) and compared differences in peptide identifications. METHODS: IgG, containing mouse and human sequence, was purchased from several vendors. Samples were prepared in Tris buffer, 0.1% RapiGest (Waters), or 6 M urea. Samples in Tris and 0.1% RapiGest were also heated to 60 °C. DTT and iodoacetamide were used to alkylate cysteine residues. Samples were digested with trypsin for 2 or 20 hours at 37 °C. Peptides were analyzed by SDS-PAGE and LC-MS/MS. Data were analyzed with NISTMSQC (NIST). RESULTS: The cysteine reduction and alkylation step was first optimized to improve alkylation and prevent protein precipitation. Urea was found to be a poor denaturant for chimeric IgG and resulted in incomplete digestion. Conversely, heating the samples improved digestions. The use of RapiGest during heating also prevented protein precipitation. Increased sequence coverage is observed in the difficult to digest CH3 region of the heavy chain upon heating. However, long digestion times result in the loss of the large, hydrophobic peptide in the CH1 region of the heavy chain. Only minor variations are observed under the different digest conditions for the light chain peptides. Therefore, heat denaturing in the presence of a surfactant followed by short digestion times were found to be optimal for chimeric IgG.