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Analysis at the Peptide Level

Typical Digestion Protocol and Enzymes

To identify, or fully characterize, a protein biopharmaceutical it must be broken down into smaller segments – peptides. This process requires proteolytic enzymes to digest the protein into peptides and is referred to as bottom-up proteomics. Great advances in chromatography and mass spectrometry have been made to obtain full structural information at the protein level (top-down proteomics), but there are still some final obstacles to overcome before this is realized as a mainstream protein analysis technique.

There is a large amount of information that can be acquired from biopharmaceutical analysis, including specific post-translational modifications (PTMs) and the protein glycoprofile (the degree and type of glycosylation).

However, this can only be isolated to specific amino acid residues when assessed at the peptide level. Great care and consideration is therefore required during the digestion process, as the proteolytic enzymes used and the conditions employed (pH, temperature, even storage time) not only affect the overall number of peptides liberated, but also the stability of associated PTMs and can even introduce protein modifications of their own. The importance of accurately assessing the true modification profile of a biopharmaceutical was illustrated when 13 different asparagine deamidations were detected, at levels in excess of 40%, when comparing the original innovator product and the trastuzumab (Herceptin) biosimilar [1]. This data also contradicted previous ion exchange data from a separate study [2]; an amendment to the study detailing issues with sample preparation was added [1].

A typical peptide mapping workflow for a monoclonal antibody (mAb) using trypsin as the proteolytic enzyme is:

Typical workflow for mAb digestion using trypsin

Figure 1: Typical workflow for mAb digestion using trypsin.

Broadly speaking the digestion process can be broken down in to three discrete and separate steps, reduction, alkylation and digestion.

The first stage in the reduction step is to denature the mAb. This is commonly accomplished with an acid labile surfactant – RapiGestTM (Waters) in this case - that acts to remove the higher order structure of the protein, and exposes many otherwise internal disulfide bonds. These disulfide bonds are then ready for reduction, which is achieved using dithiothreitol (DTT). DTT is a small molecule redox reagent, sometimes referred to as Cleland’s reagent after the biochemist who first utilized DTT as a proteolytic reducing agent. The pH is maintained at physiological levels throughout the process and buffers are used to ensure this.


To prevent reformation of disulfide bridges across the thiol groups of the cysteine (C) residues, the protein is then incubated with an alkylating agent such as 2-iodoacetamide (IAA), once again at physiological pH.


The final stage is the addition of a proteolytic agent, trypsin in this case, which is capable of  site specific protein digestion. Trypsin cleaves proteins at the C-terminal side of both lysine (Lys/K) and arginine (Arg/R) residues, unless either is proceeded by a proline (i.e. KP or RP). For this reason all resultant peptides, apart from the C-terminal peptide, terminate in either a lysine or arginine residue.
Additional and alternative proteolytic enzymes are available and are routinely used. Table 1 details these enzymes and highlights their specific cleavage sites – typically, fewer cleavage sites leads to larger, and therefore fewer, resulting peptides, and vice versa.

Enzyme Site of Cleavage
Trypsin Lys, Arg (C)
Chymotrypsin Phe, Trp, Tyr (C)
Asp-N-protease Asp, Glu (C)
Pepsin Leu, Phe, Trp, Tyr (N)
Elastase Ala, Gly, Ser (C)
Cyanogen bromide Met (C)
Endoproteinase Lys C Lys (C)

Table 1: Common proteolytic digestion enzymes and their specific cleavage sites.

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