NanoLC Chips MS/MS for the characterization of N-glycopeptides generated from trypsin digestion of a monoclonal antibody

Abstract

In the field of therapeutic recombinant proteins, monoclonal antibodies (mAbs) have achieved a rising success with more than 30 mAbs that have reached the market in the past 20 years. From a structural standpoint, one of the most important posttranslational modifications affecting antibodies is by far glycosylation. Furthermore, glycosylation of mAbs directly impacts on their biological activity and safety and therefore needs to be well characterized. Glycoprotein analysis requires high-resolution separation techniques that can provide detailed structural analysis able to discriminate between glycoforms of various abundances. This chapter describes a protocol for nanoLC-Chip-MS/MS analysis of a proteolytic digest of the heavy chain of a recombinant mAb. The use of graphitized carbon column instead of classical C18 reversed-phase material is shown to be well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.