Perfect for MS Analysis

Mass spectrometry is a key technology for identifying individual protein species in proteomics experiments. High specificity proteases digest proteins into peptides. Sophisticated software then compares actual peptide mass with the predicted mass from protein sequence information, and assigns protein identity. Trypsin cleaves peptide bonds specifically on the carboxyl side of lysine and arginine amino acid residues, creating peptides in the size range needed for mass spectrometry analysis. This cleavage specificity—and the generation of positively charged peptides, which improves ionization in the positive ion mode of the mass spectrometer—makes trypsin the enzyme of choice for MS analysis.

High Specificity

High-specificity proteases are valuable tools for MS protein identification analysis. Native trypsin is often contaminated with chymotrypsin, and trypsin autolysis creates pseudotrypsin, a derivative with broad cleavage specificity. To overcome these problems, Stratagene’s MS Grade Trypsin is modified to maximize specificity. First, porcine pancreas trypsin is reductively methylated, producing an enzyme that retains high activity and is resistant to autolysis. The modified enzyme preparation is then treated with TCPK, an irreversible inhibitor of the common contaminant chymotrypsin. Finally, the enzyme is affinity purified and lyophilized.

Table 1
Determine the Correct Protein Sequence 100 ng of carbonic anhydrase II was separated by gel electrophoresis and digested with 500 ng of MS Grade Trypsin, Trypsin Gold (Promega), or Proteomics Grade Trypsin (Sigma) overnight at 37°C. Peptides were purified as described in product manual and analyzed by MALDI-TOF MS.

Excellent Performance

MS Grade Trypsin routinely provides specific digestion of proteins in a highly efficient manner with both in-gel and in-solution protocols. As shown in Table 1, MALDI-TOF MS analysis of carbonic anhydrase resulted in positive identification with high sequence coverage (40%). Each vial of MS Grade Trypsin is lyophilized for stability and retains full activity even after five freeze-thaw cycles.

Contributing Scientists:
Stratagene: Paul Anderson, Rajesh Bagga, Ronda Allen, Dan Clark, and Jeff Braman

Internal Standards

While it is important to maximize enzyme specificity and eliminate most products of autolysis, the presence of a few well-characterized autolytic tryptic fragments is desirable for use as internal standards. Stratagene’s MS Grade Trypsin retains the ability to produce three autolytic fragments when the protease is subjected to typical reaction conditions. These three fragments—corresponding to monoisotopic masses (M+H)+ of 842.5099, 1045.5642, and 2239.1359—may be used as internal standards for mass spectrometer calibration as well as for accurate assignment of mass values to unknown peptides.