Compositional profiling of protein hydrolysates by high resolution liquid chromatography-mass spectrometry and...

Protein hydrolysates have attracted growing research and commercial attention due to their numerous nutritional, functional, and biological activities. However, only a limited range of proximate properties are determined routinely due to their substantial structural complexity and compositional variability. From both a manufacturing and functional perspective, it is of critical importance to monitor the compositional variations and identify potential similar or disparate features between different protein hydrolysates. In the current study, a single-approached method employing reverse phase ultra-high performance liquid chromatography coupled to high resolution electrospray ionization tandem mass spectrometry (RP-UHPLC-HR-ESI-MS/MS) was developed, optimized, and cross-validated for comprehensive structural and compositional profiling of a range of protein hydrolysates of varying raw materials, including soy, cotton, wheat, rice, and meat. Untargeted chemometric analysis and feature-based molecular network demonstrated potential for large-scale compositional assessment of protein hydrolysates without the need of prior component annotation. Signature features were identified to differentiate soy hydrolysates prepared from different batches of raw material and by different manufacturing processes. A hybrid approach combining de novo sequencing and target-decoy database homology search for peptide annotation is also described. Short peptides of 2 to 5 amino acids represented the most abundant components in soy protein hydrolysates (SPHs). A simple yet reliable integrated workflow for comprehensive structural and compositional profiling of protein hydrolysates was developed to enable an eventual correlation between their structure and function.