ABSTRACT: RATIONALE:Biological studies are conducted at ever-increasing rates by relying on proteomic workflows. Although data acquisition by mass spectrometry is highly automated and rapid, sample preparation continues to be the bottleneck of developing high-throughput workflows. Enzymatic protein processing, in particular, involves time-consuming protocols that can extend from one day to another. To address this gap, we developed and evaluated simple, in-solution tryptic enzymatic reactions that unfold within a few minutes, and demonstrate the utility of the methodology for the rapid analysis of proteins originating from cancer cell extracts. METHODS:Tryptic enzymatic reactions were conducted for 7-60?min, and the results were compared with that of a routine approach conducted for 18?h. No other reaction conditions were changed relative to the 18?h procedure. The reaction products were analyzed by nanospray high-performance liquid chromatography/tandem mass spectrometry (nano-HPLC/MS/MS), and the quality of the products was assessed in terms of peptide/protein identifications, sequence coverage, peptide length, missed-cleavage sites, quality of generated ions, and peptide hydrophilic/hydrophobic properties. 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.
