Protein samples are typically digested to their peptide constituents prior to peptide mapping using mass spectrometry (MS). The most frequently used enzyme for pre-MS digestion is trypsin. Our Mag-Trypsin (TPCK-trypsin immobilized on magnetic beads) allows proteins to be easily and effectively digested. After digestion, trypsin is easily removed from the final peptide mixture using a magnetic separator. The final peptide mixture is free of trypsin contaminants.
Mass Spectrometry Grade Magnetic TPCK-Trypsin Offers:
- No chymotryptic activity—prevents peptide aggregation
- No centrifugation required—complete trypsin removal with rapid magnetic separation.
- No additional mass spectrometry peaks—no trypsin contamination in your final peptide digest
- Minimal autolysis products—reduce potential interference in sample analysis caused by autolytic fragments
Immobilized TPCK-Trypsin Digestion Procedure
Proteins are effectively digested using Mag-Trypsin immobilized magnetic trypsin. When digestion is complete, Mag-Trypsin is removed from the digestion mixture using a magnetic separator— in a single step without centrifugation. The final peptide mixture is free of trypsin contaminants.
TPCK Treatment Inhibits Chymotrypsin Activity
Trypsin immobilized on magnetic beads is first treated with TPCK (a reagent reported to inhibit chymotrypsin activity without any effect on trypsin). Without TPCK treatment, chymotrypsin catalyzes the hydrolysis of peptide bonds adjacent to the carboxyl group of the aromatic amino acids tryptophan, tyrosine, and phenylalanine. Trypsin is maximally active in the pH range of 7–9 and is reversibly inactivated at pH 4.0.
Obtain a Trypsin-Free Peptide Digest
HPLC analysis of beta-casein digested with Mag-Trypsin
Denatured beta-casein protein was digested with TPCK-trypsin immobilized on magnetic beads. Reverse phase HPLC (RP-HPLC) data is shown for undigested beta-casein and digested beta-casein.
Denaturation was carried out by dissolving 1 mg of protein in 100 µl of 0.1 M sodium carbonate, pH 8.3, and 8 M urea; and mixing at room temperature for 2–6 hr. The denatured protein was then diluted to 1 ml to reduce the urea concentration to <1 M. Then 1 ml of immobilized trypsin was washed with water and incubated with the protein, with vigorous mixing at 37°C overnight. (The enzyme-to-substrate ratio and incubation time may need to be optimized for a specific application.) RP-HPLC analysis was performed on a Waters Breeze HPLC, XTerra RP18 column (5 m, 4.6 x 150 mm) with UV detection at 215 nm (Solvent A: 0.1% TFA in water [v/v], Solvent B: 0.1% TFA in acetonitrile [v/v]).
The results confirm that the immobilized trypsin is active and provides complete trypsin digestion. Thus it may be used successfully in any current techniques which utilize trypsin for protein digestion and require its removal following digestion, such as mass spectrometry or sample preparation for peptide enrichment. Mag-Trypsin also supports sample preparation for our Magnetic Phosphopeptide Enrichment Kit (Cat. No. 635643).
Free Trypsin vs. Immobilized Magnetic TPCK-Trypsin
Free trypsin, the most widely used protease for MS analysis of proteins, cleaves at lysine (lys) and arginine (arg) residues. However, there are a few disadvantages to using free trypsin:
- Longer digestion times are required
- Higher digestion temperatures are necessary
- Autolytic fragments are generated
These challenges can be overcome by using immobilized trypsin for in-solution digestion of simple or complex proteins. Magnetic TPCK-trypsin reduces digestion time and allows easy removal of trypsin from the digestion reaction using a magnetic separator—helping eliminate trypsin contamination in downstream sample analysis. It also allows flexibility and scalability by making it possible to vary the amount of resin used in the reaction.