Peptidomics and Beyond: Development of a Novel Platform Based on Capillary Electrophoresis–Mass Spectrometry for Metabolomics of Medium-Molecular-Weight Compounds

Proteomics targets proteins typically >10 000 Da, while metabolomics focuses on small molecules <1000 Da. In contrast, omics approaches targeting medium-molecular-weight compounds (MMWCs) remain underdeveloped. Although peptidomics targets these molecules, progress has been limited by several factors, including the low abundance of bioactive peptides, their instability during sample preparation, and the lack of analytical platforms capable of comprehensively analyzing diverse modified peptides and other as-yet-uncharacterized MMWCs. Here, we developed a novel high-sensitivity platform based on capillary electrophoresis coupled with high-resolution mass spectrometry (CE-HRMS) for the comprehensive analysis of peptides and other MMWCs. To achieve this, we implemented large-volume dual preconcentration by isotachophoresis and stacking (LDIS), which increased the injection volume by 60-fold over the standard volume and enabled detection limits of 10 pg/mL for peptides such as bradykinin and ANP. We then applied this novel platform to plasma samples from hyperlipidemia patients and found significantly elevated levels of a series of peptides, including bradykinin, CLIP, and schizophrenia-related peptides, along with 63 additional protein-derived peptide fragments and two unknown MMWCs. In conclusion, this platform enables systematic exploration of previously uncharacterized molecules, facilitating the discovery of novel functional peptides and other MMWC biomarkers.