Onder_2025_Chem.Biol.Interact__111695

Cohn Fraction IV-4, a byproduct of the plasma fractionation process, is a rich source of human butyrylcholinesterase (BChE). We studied the possibility that the trace quantities of acetylcholinesterase (AChE) in human plasma might be in Fraction IV-4. We searched for AChE using a multi-layered approach including immunopurification, enzyme activity assays, and high-resolution mass spectrometry. BChE was immunodepleted from an extract of frozen Cohn Fraction IV-4, leaving active AChE in the supernatant at levels similar to AChE in Silent BChE plasma (devoid of BChE). AChE was captured by a monoclonal anti-AChE antibody. Tryptic peptides of the immunopurified AChE were subjected to LC-MS/MS analysis, resulting in the identification of eight AChE peptides that clearly distinguished AChE from BChE. Erythrocyte membrane-bound AChE and full length AChE have different C-terminal sequences. Peptide LLSATDTLDEAER is unique to full length AChE. Peptide LLSATDTLDEAER was found in AChE immunopurified from plasma-derived Cohn Fraction IV-4, supporting the conclusion that AChE in plasma and AChE in Cohn Fraction IV-4 originates mainly from tissues such as brain and muscle.