Secretome profiling of antibody-producing CHO cells using non-natural amino acid crosslinking pull down and mass spectrometry

摘要

Chinese Hamster Ovary (CHO) cells are the most common hosts for manufacturing a variety of biotherapeutic drugs including monoclonal antibodies (mAbs). LC-MS2 based detection of low abundance proteins in body fluids or cell supernatants can be challenging in the presence of proteins such as albumin and immunoglobulins or when non-secreted proteins are released upon cell death. We developed a method to identify and quantify secreted proteins in a large dynamic range of protein concentrations. Herein we describe the use of azidohomoalanine (AHA), a non-natural amino acid with an azide moiety, for incorporation into newly synthesized proteins. After incorporation, newly synthesized secreted proteins were captured on a solid support and analyzed. AHA-labeled secreted proteins were also identified and quantified by label-free and TMT methods.