Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin

Abstract

Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted the number of proteins identified. Proteases with higher specificity led to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin, and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, predigesting with trypsin improves the number of identified proteins for proteinase K by 731%. Trypsin predigestion also improved the protein identifications of other proteases, AspN (+62%), GluC (+80%), and chymotrypsin (+21%). Interestingly, the sequential digest with trypsin and AspN yielded even a higher number of protein identifications than digesting with trypsin alone.

IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay

“…Another 50 μL of 50 mM ABC buffer was added to gel samples and incubated at 37 °C for overnight. After that, elastase (Promega) (150 ng/μL in water) was added to the final concentration of 7.5 ng/μL and incubated for 30 min at 37 °C (Dau et al, 2020). Digestion was stopped by the addition of 5 μL of 10% TFA.…”

Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc

“…The use of multiple proteolytic enzymes to increase proteome coverage has been reported previously [ 52 , 53 , 54 ] and a recent study showed that dual proteolytic treatment with Trypsin followed by Asp-N increased protein IDs in a yeast proteomics experiment by roughly 10% [ 55 ]. While the NGAG protocol does utilize both enzymes, the steps between these proteolysis treatments include blocking of endogenous Asp residues from Asp-N cleavage by treatment with aniline.…”

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