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 on the number of proteins identified. Proteases with higher specificity let 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, pre-digesting with trypsin improves the number of identified proteins for proteinase K by 731 %. Trypsin pre-digestion 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 higher number of protein identifications than digesting with trypsin alone.

Spatiotemporal Visualization of Cell Membrane with Amphiphilic Aggregation-Induced Emission-Active Sensor

“…To further confirm that QMC12 could real-time monitor the dynamic membrane change, trypsin (a protease used for the passage of adherent cells) was added to imitate external stimuli through changing the cell membrane morphology. 46,47 Specifically, the adherent HeLa cells were first stained with QMC12, then these cells were incubated with the trypsin to allow continuously monitoring under a confocal microscope. To our delight, the complete process of the dynamic membrane change was record clearly by using the fluorescent signal of QMC12.…”

Proteomics informed investigation of human hepatocytes and liver tissue

“…Resulting peptides are also easily further fragmented via MS. [80][81][82] To increase digestion efficiency as well as decrease sample complexity, sequential digestion is often performed. 83,84 Other ways of reducing sample complexity to increase proteome coverage is to perform fractionation, such as high pH fractionation and isoelectric focusing. 85,86…”

Mucinomics as the Next Frontier of Mass Spectrometry

“…41,42 Other proteases have been used for these purposes, such as GluC or AspN but are less common. 36,43 Suspension trapping (S-trap) can be included in the proteomic workflow, whereby spin columns with a quartz filter are used to prepare samples. In the presence of sodium dodecyl sulfate (SDS), protein mixtures are diluted and alkylated in solution.…”