The role of trypsin in bottom-up proteomics moving towards clinical application


The impact of different trypsins on commonly used bottom-up proteomics workflows were investigated across a range of sample types in a publication in Analytical Chemistry. Besides the type of trypsin, different protease concentrations, sample matrices, instrument acquisition techniques, and protease storage times were investigated.

Proteases, digesting proteins into peptides, are key components of any mass-spectrometry (MS) bottom-up proteomics experiment. Trypsin, a serine-protease produced in the pancreas of most vertebrates, is the most widely used protease due to its superior cleavage specificity and the generated peptides being of ideal size for MS and tandem MS analysis. As bottom-up MS proteomics is more frequently applied in large-scale biomedical studies and moving towards clinical applications the technical impact and possible biases in the trypsins used become of greater importance.

In this study trypsins from different vendors were tested on three different biological samples, human plasma, single HEK293 cells and HeLa cell line lysate, using different bottom-up proteomics workflows. The results show only minor differences in performance between trypsins, with the concentration having the largest effect and storage not affecting the performance.

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