Rapid Denaturing Organic Digestion Method for Targeted Protein Identification and Characterization

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