In order for a protein to be introduced into an MS for identification, it must undergo a series of preparatory steps to ensure the cleanliness of the sample. Protein digestion is among those steps. Using a protease (most commonly trypsin), the protein is enzymatically digested into smaller pieces or peptides. Laboratory automation allows scientists to automate this process, which eliminates human errors and increases throughput.

The two most commonly automated digestion methods are in-gel and in-solution digestion. For in-gel digestion, the proteins are first separated by 1D or 2D gel electrophoresis. The proteins are then picked up by a spot picker and transferred into vials and/or microplates for further cleanup. Before the proteins can be digested, a destaining step is needed to remove the dyes used during the electrophoresis process. After destaining, researchers often reduce and alkylate the proteins by using buffers. Once the proteins are digested, they must be extracted and further prepared using a special, MS-compatible buffer before introduction into an MS. The in-gel digestion method is relatively easy to automate for higher throughput and cleaner samples. However, due to the number of steps involved, significant sample loss may occur.

In-solution digestion is performed directly from a solution, and therefore many of the cleanup steps are eliminated. The clear advantage of this method is that there is little to no sample loss, leading to better detection. Consequently, in-solution digestion is most often used for samples with a scarce amount of protein. This method, however, may require further clean-up to ensure a reliable MS analysis.

Many of the vendors involved in manufacturing automated protein digestion platforms offer products designed to perform both in-gel and in-solution methods. DigiLab offers three automated systems designed for either method. The ProPrep II model adds the ability to spot directly onto MALDI plates or LC vials. Intavis’s DigestPro MSi is similar to the ProPrep II in that it is compatible with MALDI plates and can process up to 96 samples per run; however, the system is primarily used for in-gel digestion. The GE Ettan digester can handle up to eight samples simultaneously and can integrate up to 4 x 96 well plates for 384 sample digestions per batch.

Shimadzu’s new Perfinity Workstation offers a chromatographic approach to automated digestion (see page 11). The system can integrate up to five different column technologies for downstream protein separation in preparation for MS detection. It differs from other digestion methods by using immobilized trypsin in a column, which prevents autodigestion and allows the use of larger amounts of trypsin for faster digestion times.

The market for automated peptide digestion is growing due to large molecule research, particularly in biotherapeutics. Market growth is due to the push for cleaner samples, better reproducibility, faster digestion times and ease of use.

Automated Protein Digestion for LC/MS at a Glance:

Leading Suppliers

• GE Healthcare

• Shimadzu

• DigiLab

Largest Markets

• Biotechnology

• Pharmaceuticals

• Academia

Instrument Cost

• $15,000–$150,000