TECH TIPS Why Supplement Tryps Trypsin/Lys-C Mix, Mass Spec Grade, is recommended for use in two applications: enhanced standard in-solution protein digestion and in-solution digestion of proteolytically resistant proteins. Compensate for Missed Cleavage Sites and Enhance Proteolysis Efficient proteolysis is a major requirement for protein mass spectrometry analysis. Incomplete digestion has multiple ramifications including decreased number of identified proteins, compromised analytical reproducibility and poor protein quantitation. Trypsin is one of the most robust proteases, but typical trypsin reactions do not digest proteins to completion, missing 15–30% of cleavage sites. Supplementing Trypsin with Lys-C compensates for the majority of missed cleavages. 100 20 40 60 80 0 Trypsin Yeast Protein Extract 93.4% 76.8% 100 20 40 60 80 Trypsin/Lys-C Mix 100 20 40 60 80 Arabidopsis Protein Extract 94.2% 0 Trypsin Trypsin/Lys-C Mix 100 43% 20 40 60 80 FFPE Tissue Protein Extract 78.5% 91.5% K562 Protein Extract 94.7% 89.6% Side-by-side Comparison of Trypsin and Trypsin/Lys-C Digestion for Missed Cleavages (% of total cleavages). All the digests used overnight 37°C incubation. enews | Fall 2013 mpletely Digested Peptides % of Completely Digested Peptides mpletely Digested Peptides % of Completely Digested Peptides Pagina 9
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