Fig 1.
(A) The general workflow for preparation of purified, stabilized trypsin along with different parameters that were evaluated. (B) The final workflow after evaluation.
Fig 2.
Extent of trypsin methylation.
(A) MALDI-TOF spectra of intact Sigma IX-S in the non-methylated (Orange) and 60 min methylated (Blue) form. (B) The lysine content during methylation at pH 5.8 as a function of time measured by AAA. C) AAA chromatograms showing lysine and dimethyllysine in a non-methylated (left) and 60 min methylated (right) sample.
Table 1.
Purification yields.
Fig 3.
Distribution of tryptic peptides from digests.
(A) Distribution of peptide abundances (measured based on peak area) with fully tryptic (blue), tryptic-chymotryptic (gray) and semitryptic (black) peptides for a 37°C overnight digest using different trypsin preparations at 4% trypsin w/w. (B) Percentage of peptide intensities found in a digest of the simple protein mix using enhanced Sigma IX-S trypsin under different incubation times (one hour [1h] or overnight [ON]) and enzyme concentration (2 or 4% w/w trypsin-to-substrate). The plot shows the percentage of peptides with zero missed cleavages (shaded) and peptides with fully tryptic terminals (grey). (C) Distribution of peptide abundances (measured based on peak area) for a 37°C overnight digest using different trypsin preparations at 2% trypsin w/w.
Fig 4.
(A)-(D) HPLC chromatograms of trypsin during autodigestion at 37°C. Enhanced trypsin is shown after 0 (A) and 17 (C) hours. Crude Sigma IX-S trypsin is shown after 0 (B) and 17 (D) hours. (E) Stability of crude (blue diamonds) and enhanced (red squares) Sigma-IX-S trypsin towards autodigestion.
Fig 5.
Identified peptides of HeLa lysate.
Number of identified peptides with 0 missed cleavages identified after digestion of a HeLa lysate at 58°C using either crude (blue diamonds), enhanced Sigma IX-S (red squares) or PSG (green triangles) trypsin.