Introduction: Endogenous peptides play crucial roles in various biological functions and are extensively used in medical diagnostics and therapy. Although targeted analysis of individual peptides has been employed in clinical studies, a large-scale untargeted identification of endogenous plasma peptides (peptidome) by mass spectrometry is still challenging. The primary obstacle in sample preparation is the predominance of high abundant proteins, which hinders the detection of low-abundance peptide biomarkers. Additionally, the low identification rate of MS2 spectra suggests the presence of non-tryptic or modified peptides that are missed by standard methods. Our goal is to develop a robust, high-throughput workflow to improve the identification of endogenous peptides in plasma and apply it to a large clinical cohort of ischemic stroke patients, where early diagnosis is essential for tailored treatment.
Methods: We combined methanol-based precipitation to deplete abundant plasma proteins (e.g., albumin, globulins) with subsequent ultrafiltration for peptide separation (10kDa MWCO) and SCX/C18-based purification on a 96-well plate format. Peptides were analysed by Orbitrap LC-MS/MS and searched using MSFragger (ver. 4.0) with unspecific search parameters. For clinical applicability, we applied this method to plasma samples from a cohort of 24 ischemic stroke patients (collected on Day 1, 3, and 7 post-stroke) and 24 healthy controls, acquiring both proteome and peptidome data.
Results: Our optimized workflow enables the identification of over 6,000 endogenous plasma peptides from 100µl sample input, largely expanding the peptidome of previous studies. Many low abundant small proteins and peptides, such as protease inhibitors, peptide hormones, and neuropeptides were more frequently detected compared to standard proteomic analysis. Notably, in the ischemic stroke cohort, we observed a significant up-regulation of various endogenous peptides, while the proteome remained largely unaffected. Many of these peptides were derived from proteins associated with the coagulation cascade, such as Fibrinogen (A and B), Coagulation Factor IX, and Serum Amyloid A1. Mapping the peptidome to the sequence localization of the underlying proteins revealed a preference for specific peptide termini, suggesting stroke-specific cleavage events possibly induced during the acute phase.
Conclusions: Our peptidome workflow facilitates the untargeted identification of the largest number of endogenous peptides in plasma to date, suitable for large clinical cohorts in the 96-well format, and easily transferable to other biofluids. Complementary to proteome measurement, peptidome profiling reveals disease-specific proteolysis in clinical samples, serving as a potential biomarker signature.