Strategies for mapping protein hydrolysate profiles and pharmacokinetics based on non-targeted proteomics combining skyline-aided quantitative techniques

Numerous works have been focused on the bioactivities of protein hydrolysates (PHs) and their application in food or drug formulations, but their composition and pharmacokinetics have never been addressed due to their complex constitutes, short half-life, extremely low concentrations and lack of authentic standards. The present study aims to develop systematic analytical strategy and technical platform with optimized sample preparation, separation and detection protocols for PHs. Lineal peptides (LPs), extraction of the spleen of healthy pigs or calves, were used as cases. First, solvents with polarity gradients were used to globally extract peptides of LP from biological matrix. Non-targeted proteomics based on a high-resolution MS system was used to establish a reliable qualitative analysis workflow for PHs. Based on the developed approach, 247 unique peptides were identified using NanoLC-Orbitrap-MS/MS, and then further verified on the MicroLC-Q-TOF/MS system. In the quantitative analysis workflow, Skyline software was used to predict and optimize the LC-MS/MS detection parameters of LPs followed by investigating the linearity and precision of the developed analytical assay. Note worthily, we innovatively prepared calibration curves by sequential dilution of LP solution to overcome the bottleneck of lacking authentic standards and complex PH composition. All the peptides exhibited good linearity and precision in biological matrix. The established qualitative and quantitative assays were successfully applied to study the distribution characteristics of LPs in mice, and would be conductive to systematically map the profile and pharmacokinetics of peptides in various PHs in vivo and in vitro.