Simultaneous extraction and analysis of multiple cystine-dense peptides by μSPE and microflow-MS/MS from plasma

Aim: Develop a universal extraction and liquid chromatography-mass spectrometer method to simultaneously analyze cystine-dense peptide (CDP) miniproteins from rat and human plasma. The results of the analysis will be used to assist selection of therapeutic drug candidates from the vast CDP library. Methods & results: A micro-elution solid-phase extraction method was developed for the sample preparation of the CDP peptides in rat and human plasma followed by analysis by microflow liquid chromatography MS/MS. The methods developed for drug discovery were found to be accurate (±≤15.2% from nominal concentrations) and precise (≤13.4% CV), with a dynamic range of 1.00–500 ng/ml and extraction recoveries of 47.2–99.0%. Conclusion: This bioanalytical method can be utilized to screen CDP proteins and other miniproteins for drug discovery, candidate selection and further drug development.