616204-83-2Relevant academic research and scientific papers
Thermally enhanced enzymatic proteolysis for rapid 18O labeling in proteomics
Antoine, Miquel D.,Hagan, Nathan A.,Demirev, Plamen A.
experimental part, p. 24 - 29 (2012/06/18)
To streamline protocols for protein identification and better understand the contributions of purely thermal versus non-thermal effects, we utilize a microwave oven and a PCR thermocycler for rapid heating to accelerate enzymatic digestion of proteins. When performed in H218O, rapid heating results in efficient C-terminal 18O atom labeling of the proteolytic peptides. The approach is illustrated on the example of several pure proteins using trypsin and other proteases for rapid digestion. MALDI TOF/TOF MS provides unambiguous identification of the individual tryptic peptides. We have performed a time-course study on the degree of 18O incorporation by varying the irradiation/heating times for each method. In order to gain insights into the mechanism of thermally enhanced trypsin digestion and 18O labeling we carry out experiments in which the two events - lysis and labeling - are decoupled. We also study the rates of 18O incorporation as a function of tryptic peptide C-terminal amino acid type and peptide length. Both heating methods are very rapid - in most cases digestion and incorporation of two 18O atoms into R-terminated tryptic peptides is completed in less than 5 min, thus considerably reducing the time for bottom-up proteomics including quantitation by 18O labeling.
