Conclusions
We demonstrate that partial enzymatic reactions conducted on short time-scales represent a valuable asset to proteomic studies, and propose their implementation either as simple, cost-effective, stand-alone protocols for substantially streamlining the analysis of biological samples, or as complementary protocols, for improving protein sequence and proteome coverage.
Methods
Tryptic enzymatic reactions were conducted for 7-60 min, and the
Results
The results demonstrate that brief, and therefore incomplete, enzymatic processes lead to a large number of peptide fragments that improve protein sequence and proteome coverage, that the tandem mass spectra produced from these peptides are of high quality for reliable protein identifications, and that the physical properties of peptides are prone to supporting the development of alternative multi-dimensional separations and middle-down proteomics analysis strategies. The reproducibility of generating the same peptides within a few minutes of enzymatic digestion was remarkably close to that obtained from 18 h long reactions, and the combined results of short and long reactions increased proteome coverage by ~40%. Conclusions: We demonstrate that partial enzymatic reactions conducted on short time-scales represent a valuable asset to proteomic studies, and propose their implementation either as simple, cost-effective, stand-alone protocols for substantially streamlining the analysis of biological samples, or as complementary protocols, for improving protein sequence and proteome coverage.