A new sample preparation method for the absolute quantitation of a target proteome using (18)O labeling combined with multiple reaction monitoring mass spectrometry.

A key step in the workflow of bottom-up proteomics is the proteolysis of proteins into peptides with trypsin. In addition, enzyme-catalytic (18)O labeled peptides as internal standards coupled with multiple reaction monitoring mass spectrometry (MRM MS) for the absolute quantitation of the target proteome is commonly used for its convenient operation and low cost. However, long digestion and labeling times, incomplete digestion and (18)O to (16)O back exchange limit its application, therefore, we developed a rapid and efficient digestion method based on a high ratio of trypsin to protein. In addition, after separation of the digested samples using pipette tips packed with reversed-phase packing materials in house, the trypsin can be separated, collected and reused at least four times. Based on this approach, a novel protein quantification method using (18)O-labeled QconCAT peptides as internal standards combined with MRM MS for the absolute quantitation of a target proteome is established. Experimental results showed that the novel method had high digestion and (18)O labeling efficiencies, and no (18)O to (16)O back-exchange occurred. A linear range covering 2 orders of magnitude and a limit of quantification (LOQ) as low as 5 fmol were achieved with an RSD below 10%. Then, the quantitative method is used for the absolute quantitation of drug metabolizing enzymes in human liver microsomes. The results are in good agreement with the previously reported data, which demonstrates that the novel method can be used for absolute quantitative analyses of target proteomes in complex biological samples.