Thermally enhanced enzymatic proteolysis for rapid 18O labeling in proteomics

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.

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► We streamline protein identification protocols utilizing a microwave oven and a PCR thermocycler to accelerate enzymatic digestion.
► Performed in H218O, rapid heating results in efficient C-terminal 18O atom labeling of the proteolytic peptides.
► We investigate rates of 18O incorporation as a function of tryptic peptide C-terminal amino acid type and peptide length.
► In most cases, digestion and incorporation of two 18O atoms into R-terminated tryptic peptides is completed in less than five minutes.