A Novel Method for Creating a Synthetic L-DOPA Proteome and In Vitro Evidence of Incorporation

Abstract

Proteinopathies are protein misfolding diseases that have an underlying factor that affects the conformation of proteoforms. A factor hypothesised to play a role in these diseases is the incorporation of non-protein amino acids into proteins, with a key example being the therapeutic drug levodopa. The presence of levodopa as a protein constituent has been explored in several studies, but it has not been examined in a global proteomic manner. This paper provides a proof-of-concept method for enzymatically creating levodopa-containing proteins using the enzyme tyrosinase and provides spectral evidence of in vitro incorporation in addition to the induction of the unfolded protein response due to levodopa.

Keywords: L-DOPA; Levodopa; PTM; misincorporation; post-translational modifications.

Figure 1

Structural forms of phenylalanine, para-tyrosine, meta-tyrosine, ortho-tyrosine, and L-DOPA. Hydroxyl radical attack on the phenyl ring of phenylalanine residues can form isomers of tyrosine, the canonical form being para-tyrosine, but can also form meta-tyrosine and ortho-tyrosine; hydroxyl attack of tyrosine can also form L-DOPA.

Figure 2

Spectral evidence of a positive control for PB-DOPA. (A) The native peptide sequence. (B) Addition of oxygen to tyrosine indicated by the increase in peptide mass and altered masses of the b8 and y10 ions.

Figure 3

The overall oxidation profile of untreated, DOPA-treated and converted SH-SY5Y proteomes. The oxidation profile of methionine, tyrosine (L-DOPA), and phenylalanine is shown for each treatment group. The level of oxidation is presented as a percentage of the total number of identified peptides containing M, Y or F (top) and individual residues (bottom). The three biological control samples were pooled for the tyrosinase conversion, using tyrosinase to peptide digest ratios of 10:1, 2:1, and 1:1. Regardless of residue or peptide sequence, the percentage of oxidation follows a similar pattern for each sample. Error bars represent the standard error of the mean (SEM).

Figure 4

The ratio of total ion intensities compared to the control. Error bars represent the SEM.

Figure 5

Volcano plot of proteins from label-free quantitative analysis. Coloured proteins have p-value <0.05 and a log₂ fold change of ≥1. Blue proteins are up in treatment, and red proteins are down.