A novel approach to detect the presence of levodopa (lDOPA) in the polypeptide chains of proteins

Background: Deposition of protein aggregates in neurons is a hallmark of neurodegenerative diseases. Genetic mutations in proteins can result in the synthesis of non-native proteins that accumulate in cystosolic aggregates. Certain non-protein amino acids (NPAAs) can be mischarged onto tRNA and mistakenly inserted into the polypeptide chain of proteins resulting in the sporadic generation of non-native misfolded proteins [1,2]. We utilise a novel approach to investigate the ability of the therapeutic agent levodopa (L-DOPA) to replace L-tyrosine in proteins by applying mass spectrometry (MS) whereby we can sequence peptides to reveal the presence of DOPA.
Methods: We examine the ability of high resolution accurate mass mass-spectrometry (HRAM-MS) to detect DOPA in neuronal cell proteins avoiding the need for traditional protein hydrolysis and to allow individual proteins to be identified.
Results: We successfully tested a novel approach for the detection of DOPA in peptides generated from digestion of DOPA-containing proteins and validated this method using synthetic peptides. We showed that L-DOPA can be incorporated into cell proteins. The presence of L-DOPA in proteins resulted in a decrease in solubility. Specific structural and long-lived proteins were most affected. We are now applying this approach to biological samples.