Errors in peptide synthesis are a source of discrepancies in Aβ42 studies

ABSTRACT Amyloid-β42 (Aβ42) aggregation is highly sensitive to experimental conditions, making reproducibility a persistent challenge in Alzheimer’s disease research. Among the many variables that influence aggregation, the impact of peptide production remains poorly understood. Direct comparison of recombinant and chemically synthesised Aβ42 prepared under carefully controlled conditions reveals that, despite following similar aggregation mechanism and forming the same predominant fibril structures, synthetic Aβ42 aggregates more slowly and exhibits reduced seeding efficiency. Consequently, synthetic Aβ42 produces fewer oligomeric species and displays lower cellular toxicity. Mass spectrometric analyses identify low-abundance sequence imperfections introduced during peptide synthesis as the origin of these differences. By linking synthesis-derived imperfections to variations in Aβ42 behaviour, this work reveals a previously underappreciated source of discrepancies in amyloid studies. In addition, we provide a framework for evaluating the impact of sequence impurities on biophysical studies that are sensitive to peptide composition.