Capturem Pepsin was developed for rapid and efficient digestion of antibodies—with a room-temperature protocol (Figure 1)—designed for downstream proteomics analysis. Pepsin is an acidic protease with utility in a wide variety of applications, most notably in the preparation of antibody samples for characterization by mass spectrometry (MS). The specificity (Keil, B. et al. 1992) and activity of digestion are both pH-dependent, with an active range between pH 1–5. For the purpose of MS, digestions are typically carried out at pH 1–2 in order to cleave peptide bonds at both N- and C-terminal phenylalanine, leucine, tryptophan, and tyrosine residues. Altering the pH of the reaction will, in turn, affect the amount of digestion that occurs; 90% of maximum activity is achieved at pH 1.5, while only 35% activity is achieved at pH 4.5 (Bohak, Z. et al. 1969).
Conventional methods for sample preparation involve in-solution digestion with pepsin, in which incubation times can be as long as overnight. In addition to being a time-consuming process, such a prolonged digestion may lead to protein modifications, poor specificity, and over-digestion. On the other hand, shorter incubations can lead to incomplete digestion and thus compromise downstream efforts to characterize the protein. Capturem Pepsin is part of the ever-expanding Capturem family and takes advantage of this novel technology, modified here to provide membrane-immobilized pepsin in a mini spin column format. This new method offers high performance with an incredibly simple workflow, and maintains full digestion capabilities in the presence of common antibody sample prep reagents (e.g., TCEP, urea, formic acid, etc.). These single-use, disposable spin columns allow for complete digestion of antibody samples in just three minutes, providing reliable, consistent results that outperform those of lengthier in-solution methods.