Rapid denaturing organic digestion method for targeted protein identification and characterization

Oyler, J.M., Tran, B.Q. and Kilgour, D.P.A. ORCID: 0000-0002-3860-7532, 2021. Rapid denaturing organic digestion method for targeted protein identification and characterization. Analytical Chemistry, 93 (12), pp. 5046-5053. ISSN 0003-2700

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Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18–24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A Clostridium botulinum toxin, Staphylococcus enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.

Item Type: Journal article
Publication Title: Analytical Chemistry
Creators: Oyler, J.M., Tran, B.Q. and Kilgour, D.P.A.
Publisher: American Chemical Society
Date: 30 March 2021
Volume: 93
Number: 12
ISSN: 0003-2700
Divisions: Schools > School of Science and Technology
Record created by: Lee Houghton
Date Added: 22 Mar 2021 14:19
Last Modified: 17 Mar 2022 03:00
URI: https://irep.ntu.ac.uk/id/eprint/42560

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