Trypsin under the microscope

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  • Published: Aug 20, 2014
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics

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The use of trypsin for digesting proteins in proteomics studies has become so routine now that many research groups follow the standard protocols for in-gel and in-solution digestions. They work, so why bother tweaking them?

Well, two recent reports have shown that tweaking can actually provide better results and better protein coverage. One of my recent ezines described how reducing the concentration of trypsin by at least 10-fold for in-gel digestion reduced the number of self-digestion products and increased the number of proteins that could be identified. Of course, it also saves money by reducing enzyme consumption.

Now, a second report from scientists at the University of Oslo in Proteomics has explained how in-solution digestion of protein mixtures can be improved by diverging from the recognised protocol. They showed that conventional digestion times of several hours produce a significant number of peptides of molecular mass lower than 700 Da, which are difficult to detect by LC/MS.

Their alternative procedure employed short digestion times of a few minutes, which had the effect of producing larger peptides which carry more of the protein sequence. The rapid digestion also gave more reproducible ion intensities than the long process, which could be improved further by automation. They recommended that digestion should not be allowed to go to completion because so much information is lost in the small peptides.

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