Rapid trypsin digestion at elevated temperature with calcium for enhanced bottom-up proteome analysis

Trypsin is frequently employed to cleave proteins ahead of mass spectrometry characterization. Traditionally, enzyme digestion involves overnight incubation of proteins at 37 °C, which is not only time consuming but can still yield in poor digestion efficiency. While raising the temperature should theoretically accelerate the digestion, this also destabilizes the enzyme and enhances trypsin degradation. We therefore questioned whether elevated temperature digestion is beneficial for improving tryptic digestion. Here we quantify protein digestion kinetics at elevated temperatures for calcium-stabilized trypsin and enforce the critical importance of calcium ions to preserve the enzyme. We quantitatively demonstrate that 1 hour at 47 °C provides a superior digest when compared to conventional (overnight, 37 °C) processing of the proteome. The impact of our enhanced digestion protocol is shown through bottom-up mass spectrometry analysis of a complex proteome mixture.