Peptidomic analysis of cerebrospinal fluid samples reveals new biomarker candidates for amyotrophic lateral sclerosis

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating and progressive neurodegenerative disease that affects motor neurons and leading to death within 3 years. Diagnosis is based on clinical symptoms, the pathophysiology is still unclear for most patients and treatment options are limited. Therefore, there is an urgent need for fluid biomarkers to support basic research, diagnosis and drug development in ALS. Methods: We applied mass-spectrometry-based peptidomic analysis in cerebrospinal fluid (CSF) samples of ALS and non-neurodegenerative control patients (Con) from a discovery (24 ALS, 24 Con) and validation cohort of patients (67 ALS, 42 Con). Peptides were isolated and purified from CSF for unbiased peptidomics in the discovery cohort and analyzed via mass spectrometry (MS). Selected peptide candidates were evaluated by targeted parallel reaction monitoring (PRM) in the validation cohort including stable isotope-labelled peptides. Analyses were performed with an UltiMate 3000 RSLCnano system and an Orbitrap Exploris 480 MS equipped with FAIMS interface. Screening data were processed with the PEAKS software and targeted PRM data were analyzed in Skyline. Peptide abundance changes in the validation cohort were correlated with clinical parameters. Results: We identified 33605 peptides in CSF samples from the discovery cohort and 56 of them were significantly regulated in ALS compared to controls. A coefficient of variation (CV) of 6.1% was obtained for the number of identified peptides over all patients and the abundance dynamic range of peptides in patient samples spanned eight orders of magnitude. Systematic selection for the best candidates revealed a targeted PRM method with 8 peptides, which was validated in terms of intraassay variation (CV 5-12%), dilution stability (accuracy 80-119%) and freeze-thaw stability (accuracy 89-148%). The targeted PRM confirmed the abundance changes of the discovery experiment for all peptides. Combination of all peptides in a logistic regression model led to an area under the curve value of 98.4% for the discrimination of ALS from controls. PRM data of the neurofilament light chain (NfL) peptide strongly correlated with an established NfL immunoassay (Ella) (r=0.968, n=109). Conclusion: We report eight peptides derived from seven proteins as biomarker candidates for ALS. The peptides are derived from proteins with different function and their determination with our PRM method provides the opportunity for simultaneous investigation of key processes in ALS and other neurodegenerative diseases (neurodegeneration, metabolic alteration, muscle weakness/atrophy and synaptic function or the secretory pathway). Future studies must clarify their clinical value for diagnosis, monitoring disease progression and treatment response, their changes in the preclinical phase of ALS and whether the peptide level changes are indicative of the parent protein concentration or reflect changes in protein processing.