Question for my proteomics skywalkers: today I was chatting about enzymes that aren’t trypsin, and I know there’s plenty out there (including this fun paper, h/t @pastelbio.bsky.social), but any great ones that should be on my radar for bottom-up proteomics?
https://pubs.acs.org/doi/10.1021/acs.analchem.4c04277
https://pubs.acs.org/doi/10.1021/acs.analchem.4c04277
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https://pubs.acs.org/doi/10.1021/jasms.3c00448
https://www.nature.com/articles/nprot.2016.057
https://doi.org/10.1111/febs.15190
And
https://doi.org/10.1016/j.celrep.2015.05.029
https://pubs.acs.org/doi/10.1021/acs.analchem.4c04277
• Cleavage specificity: Prefers aromatic residues (Phe, Tyr, Trp) and sometimes Leu.
• Why use it?: Complementary to trypsin, it produces peptides that can help increase sequence coverage and improve protein identification in complex samples.
• Cleavage specificity: C-terminal to Glu and, less commonly, Asp.
• Why use it?: Ideal for targeting acidic residues, useful for mapping post-translational modifications (PTMs) like phosphorylation or in proteins with domains rich in acidic residues.
• Cleavage specificity: C-terminal to Lys.
• Why use it?: Often used in tandem with trypsin for increased digestion efficiency or when proteins are resistant to trypsin alone. It generates longer peptides for deeper sequence coverage.
• Cleavage specificity: N-terminal to Asp (and occasionally Glu).
• Why use it?: Particularly useful for analyzing proteins with abundant Asp residues or for PTM studies, offering orthogonal cleavage patterns to trypsin.
• Cleavage specificity: C-terminal to Arg.
• Why use it?: Produces peptides complementary to those generated by trypsin. Helpful when you want to avoid cleaving after Lys or for proteins enriched in Arg residues.
https://www.nature.com/articles/s41586-023-06921-9
Still, if you need to focus on a specific site, trypsin will not always do the job.
Worthington Glu C works well - you can order a mg of the enzyme at not extortionate prices!
We also did a comparison of expensive GluC vs. suspiciously cheap GluC for some targeted work (can't recall if it was the Worthington one) and it does make a difference w.r.t. reproducibility and efficiency. Tl;dr, you generally get what you pay for.
https://pubmed.ncbi.nlm.nih.gov/29972309/