on 01-08-201302:37 AM - edited on 10-15-202112:06 PM by Closed Account
Lassahn PG, Scheffler K, Demant M, Delmotte N, Wagner-Redeker W, Boehm G. Poster Note The quantitation of proteins in serum is essential for the assessment of the immune response to therapeutic proteins such as monoclonal antibodies and for understanding the immunological properties of these proteins. If therapeutic and endogenous proteins have to be distinguished or several proteins need to be monitored in parallel the use of ligand binding assays such as ELISAs is limited. Liquid chromatography coupled with mass spectrometry (LC-MS) is an emerging technology for this application. Whereas triple quad type mass spectrometers have been the instrument of choice for high throughput quantitation assays in the past, instruments providing high resolution and accurate mass have started to show their benefits in this application area.
Here we have developed a rapid and robust quantitation strategy of Cetuximab (Erbitux, a therapeutic monoclonal antibody for the treatment of colorectal cancer) and two additional protein standards (bovine serum albumin and bovine transferrin) spiked into human plasma with a LLOQ at the low pmol/mL level. Following the addition of isotope labeled standard peptides as internal standards, the plasma samples were reduced, alkylated and digested with trypsin. One of the key challenges in the sample preparation workflow, the control of the digestion step, was addressed by optimizing digest conditions such as time, temperature and amount of trypsin used. Samples were analyzed using a high resolution non-targeted full-scan-MS method in comparison with a targeted SIM and targeted MS/MS methods selectively focusing on proteotypic peptides and their corresponding isotope labeled analogs.