Proteins are typically measured in positive ion mode, with the charge produced on the protein originating from protonation of basic residues; arginine, lysine, histidine, and the N-terminus. With protein level analysis, it is the intact protein, or large fragments thereof, being analyzed. In the case of monoclonal antibodies, the light and heavy chains are large fragments, generated by disulfide bond reduction, the Fab and Fc fragments are generated by papain cleavage, and the F(ab’)2 fragment by pepsin or immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS) cleavage. These slightly smaller fragments are more amenable to chromatographic and mass spectrometric analysis, making the identification of particular modifications at specific domains easier. This module will explore the different techniques used for protein level analysis and look at the different information provided by each technique.

Topics include:

• Determining molecular weight of an intact protein
• Reverse phase HPLC and MS detection
• Ion exchange chromatography and MS detection
• Size exclusion chromatography and MS detection
• Hydrophobic interaction chromatography and MS detection
• MS/MS at the protein level