Orbitrap Illustration

Bottom Up Protein Characterization

Since synthetic human insulin was first introduced in 1982, the use of peptides or proteins as pharmaceuticals has steadily gained momentum and today rivals that of small-molecule-based pharmaceuticals.

As therapeutics, proteins have advantages over small-molecule drugs: they are highly specific and generally have fewer side effects. Although the primary sequence of a protein drug is homogeneous, all therapeutic proteins produced from current technologies have heterogeneities in the final product because various modifications occur during different stages of production.  Due to these heterogeneities, thorough structure characterization is necessary for the reproducible and safe production of therapeutic proteins. Complete characterization of the quality attributes of these molecules requires sophisticated techniques.

Mass spectrometry (MS), especially high-resolution and accurate-mass MS, has become widely used in virtually all phases of protein drug development.  Modern MS technologies provide accurate information about various protein properties, such as intact molecular mass, amino acid sequence, post-translational modifications like stoichiometry of glycosylated isoforms, and impurities due to sample processing and storage, as well as higher-order structural information, such as protein conformational changes upon modifications, noncovalent interactions between protein drugs and receptor proteins, and protein aggregation caused by misfolding.

For primary structural analysis, the amino acid sequence of any protein-based human therapeutics must be fully confirmed. Modifications and impurities also need to be characterized at the residue level. Usually a bottom-up approach is used in which the protein is digested by a protease, such as trypsin, into a mixture of peptides before MS analysis. The peptide mixture is separated by liquid chromatography and introduced into a mass spectrometer using electrospray ionization.

Click the links below to view the the basic LC-MS/MS bottom-up experiment for sequence analysis and post-translational modification characterization of proteins.