Overview
Purpose: To improve the efficiency and throughput of a tryptic digestion of Klotho, a highly glycosylated membrane protein.

Methods: Recombinant Mouse Klotho (R&D Sciences 1819-KL-050) was reduced,
alkylated and digested with trypsin using various methods, including Pressure Cycling
Technology (PCT). Heavy isotope labeled internal standards allowed for the
determination of absolute quantitation and digestion efficiency, using LC-SRM on a
TSQ Vantage. Samples were also run on the Exactive mass spectrometer in order to
determine how non-targeted peptides behaved with different digestion methods.
Results: PCT can significantly improve the efficiency and throughput of a tryptic
digestion when monitoring the FSISWAR peptide of Recombinant Mouse Klotho.