Proteomic interrogation of complex biomedical samples using the rapid denaturing organic digestion (DOD) method

Limitations to many current aqueous-based tryptic digestion methods include lengthy digestion times and both relatively high inter- and intra-day variability for both characteristic peptides identified and sequence coverages. This report describes results from digestion of some complex biomedical samples using the rapid Denaturing Organic Digestion method (DOD), an organic solvent-modified digestion method previously optimized for targeted protein digestion. Advantages of the DOD method included a very rapid digestion only requiring inexpensive solvents and reagents generally available in the laboratory, with no requirement for specialized equipment or expensive, specialized consumables. For this study, samples of E. coli and murine ileum protein extracts, and K562, a mass spectrometry-compatible human protein extract and reference standard routinely used to evaluate methods, were digested. Sequence coverage and characteristic peptide identification results were compared to those from 18 and 24 h conventional aqueous-based digestion methods. Across the samples tested, though the number of characteristic peptides and sequence coverages produced by the 5 min DOD method were very similar to those produced by the aqueous-based digestion methods, the specific characteristic proteins and their corresponding tryptic peptides identified following DOD method digestion included more hydrophilic and less hydrophobic species. In addition, we explored the effect of increasing digestion times with complex samples from 5 to 30 and 90 min for the DOD method. Increasing the digestion time to ≥30 min resulted in improved intra-day precision and the identification of many more peptide products than the currently used aqueous methods to which it was compared. These results suggest that the DOD organic-modified digestion method could, while markedly reducing protein digestion time, also provide more precise analysis and access to a somewhat different area of the proteome than that provided by current aqueous-based digestion methods.

Significance

The DOD tryptic digest method is a very simple and rapid process with no requirement for expensive equipment or consumables. The method markedly reduces tryptic digestion time and cost, and substantially improves within-batch and across-analyst precision for peptide and sequence coverage results over methods to which it was compared. Importantly, it also provides access to a somewhat different subset of the proteome with different peptide products identified as compared to aqueous solvent-based digestion providing potential for increased proteome coverage for bottom-up analysis if used in conjunction with aqueous-based methods.