Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides

ABSTRACT: Chronic low dose exposure to organophosphorus pesticides is associated with risk of neurodegenerative disease. The mechanism of neurotoxicity is unknown, though it is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 x 10-‐8 M) were determined. Cultured human neuroblastoma SH-‐SY5Y and mouse neuroblastoma N2a cells incubated with a sub-‐cytotoxic dose of 10 µM chlorpyrifos oxon contained depY-‐modified proteins detected by monoclonal 1C6 on Western blots. DepY-labeled peptides from tryptic digests of cell lysates were imunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile, 1% formic acid were analyzed by LC-‐MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in human SH-‐SY5Y cell lysate and 73 diethoxyphosphate-‐modified peptides in mouse N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low dose exposure t organophosphorus pesticides.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Lawrence Schopfer  

LAB HEAD: Oksana Lockridge

PROVIDER: PXD027203 | Pride | 2022-02-17

REPOSITORIES: Pride