Background: The bottom-up proteomic approach predominantly relies on trypsin or Lys-C/trypsin digestion to convert proteins into peptides, which are then analyzed by LC-MS/MS. Typically, proteomic results are derived from overnight digestions at 37°C, considered the optimal temperature for trypsin activity. However, we previously discovered that overnight urea in-solution Lys-C/trypsin digestion at room temperature (RT) outperforms the classical digestion at 37°C (1). This method not only reduces carbamylation at α- and ε-amino groups but also enhances the number and relative intensity of peptides and proteins. Additionally, it demonstrates higher reproducibility and less formation of other post-translational modifications (PTMs) such as pyroglutamic acid. Building on these results, we compared the performance of several samples preparation methods when the enzymatic digestions were carried out at 37°C and at room temperature.

Materials and Methods: BT-549 cell lysates and plasma samples were processed and analyzed in triplicates using five different approaches: Urea- and GuCl in-solution digestion, FASP, MED-FASP, and S-trap methods, with both 37°C and room temperature overnight digestions. Peptide mixtures were separated and analyzed using an Evosep One LC system (Evosep Biosystems) coupled to a timsTOF Pro ion mobility mass spectrometer (Bruker Daltonics). MS data was acquired using the 60 samples per day DD-PASEF method. Database searches were performed using FragPipe (v20.0). False-discovery rate (FDR) was filtered at 1% at both the precursor and protein levels. All statistical analyses were conducted in Perseus (v.1.6.13.0)

Results: Our study indicates that, regardless of the sample type and preparation method, overnight enzymatic digestion at room temperature with trypsin, Lys-C or Lys-C/trypsin outperforms digestion at 37°C. This was consistently demonstrated by an increase in the number of identified peptides (up to 32%) and proteins (up to 16%), alongside higher peptide signal intensities, a significant number of proteins with higher abundance (70-80%), and comparable or lower coefficients of variation for quantified proteins. These advantages come with only a slight increase (up to 4%) in the number of missed cleaved peptides. To uncover the underlying causes of these results, we evaluated several parameters, including nonspecific cleavages, proteome physico-chemical properties, and protease enzymatic activities. Our findings suggest that the significantly reduced autolysis of trypsin and Lys-C is a crucial factor underlying the enhanced efficiency of overnight digestions at room temperature.

Conclusion: This study provides strong evidence supporting the use of room temperature for all overnight enzymatic digestions with trypsin, Lys-C, or Lys-C/trypsin, given its superior performance across several methods and samples.

(1) J. Proteome Res. 2018, 17, 2556−2561.