Efficient digestion of proteins is needed for reproducible identification and quantitation in mass spectrometry. Trypsin is the gold standard enzyme for shotgun proteomics but does not efficiently digest formalin-fixed paraffin-embedded (FFPE) tissues due to protein crosslinking. Commercial sources of trypsin have been chemically modified to enhance enzymatic efficiency, avoid autolytic digestion, and suppress chymotryptic activity, but have not been compared.
We compared three sources of trypsin to digest FFPE rat kidney samples using two methods - bench top digestion and an in-house pressure-cycling technology method. Ten μm sections were lysed, reduced, and alkylated in sodium deoxycholate buffer. For bench top digestion, 2 μg of trypsin was used and samples incubated according to the manufacturer protocols. For the pressure-cycling technology digestion, 1 μg was used and the samples were digested in a barocycler using a 30-minute in-house protocol. Digests were cleaned on Waters Oasis HLB cartridges, separated by microflow chromatography, and analysed in Information Dependent Acquisition mode on a TripleTOF 6600. Digestion efficiency was determined using Protein Pilot and the Paragon Algorithm.
For the bench top method, the tryptic digestion efficiency was highest using trypsin A (83%, cf 72% and 69% for trypsins B and C). For the pressure-cycling method, trypsin C produced the highest digestion efficiency (83%, cf 66% and 47% for trypsins A and B). For both methods, the main gap was under-cleaved peptides, with a minor 3-5% over-cleaved and 1% a mixture of over- and under-cleaved. Samples with higher digestion efficiencies showed a parallel increase in peptide identifications and the percentage of peptides with a +2 charge state. Peptide identifications were up to 42% higher in samples digested using pressure-cycling technology compared to benchtop methods. Therefore, the commercial source of stabilised trypsin and experimental protocol has a significant impact on digestion efficiency and peptide identification.