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

Oyler, J ORCID: https://orcid.org/0000-0003-4427-6597, Sullivan, RF, Tran, BQ, Baker, D, Coveney, C ORCID: https://orcid.org/0000-0001-7047-6408, Boocock, D ORCID: https://orcid.org/0000-0002-7333-3549, Oyler, B, Perry, CC ORCID: https://orcid.org/0000-0003-1517-468X and Kilgour, DPA, 2025. Proteomic interrogation of complex biomedical samples using the rapid denaturing organic digestion (DOD) method. Journal of Proteomics, 312: 105359. ISSN 1874-3919

Text 2315258_Perry.pdf - Post-print Full-text access embargoed until 28 November 2025. Download (814kB)

Abstract

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.

Item Type:	Journal article
Publication Title:	Journal of Proteomics
Creators:	Oyler, J., Sullivan, R.F., Tran, B.Q., Baker, D., Coveney, C., Boocock, D., Oyler, B., Perry, C.C. and Kilgour, D.P.A.
Publisher:	Elsevier
Date:	February 2025
Volume:	312
ISSN:	1874-3919
Identifiers:	Number Type 10.1016/j.jprot.2024.105359 DOI S1874391924002914 Publisher Item Identifier 2315258 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Jonathan Gallacher
Date Added:	24 Feb 2025 09:11
Last Modified:	24 Feb 2025 09:11
URI:	https://irep.ntu.ac.uk/id/eprint/53109

Actions (login required)

Edit View

Statistics

Views

Downloads