proteomes Article A Novel Method for Creating a Synthetic L-DOPA Proteome and In Vitro Evidence of Incorporation Joel Ricky Steele 1,2,* , Natalie Strange 3 , Kenneth J. Rodgers 2 and Matthew P. Padula 1 1 Proteomics Core Facility and School of Life Sciences, The University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] 2 Neurotoxin Research Group, School of Life Sciences, The University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] 3 School of Life Sciences, The University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] * Correspondence: [email protected] 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: misincorporation; post-translational modifications; PTM; Levodopa; L-DOPA Citation: Steele, J.R.; Strange, N.; Rodgers, K.J.; Padula, M.P. A Novel Method for Creating a Synthetic L-DOPA Proteome and In Vitro 1. Introduction Evidence of Incorporation. Proteomes 2021, 9, 24. https://doi.org/10.3390/ The non-protein amino acid (NPAA) L-3,4-dihydroxyphenylalanine (L-DOPA), com- proteomes9020024 mercially known as ‘levodopa’, is used to restore dopamine levels in the damaged substan- tia nigra of Parkinson’s disease (PD) patients [1] although it has also been hypothesised to Academic Editors: Jacek accelerate PD pathology and neurodegeneration [2] by causing neuronal toxicity [2–16]. R. Wisniewski and Gunnar Dittmar The mechanisms of L-DOPA-induced neurodegeneration include induction of oxidative stress and the misincorporation of L-DOPA into proteins in place of L-tyrosine, resulting in Received: 19 March 2021 protein misfolding and the formation of proteolysis-resistant aggregates [3,5,7,17–19]. To Accepted: 18 May 2021 date, in vitro studies have not examined or identified the proteins containing incorporated Published: 24 May 2021 L-DOPA and their potential role in disease pathology, with Parkinson’s disease datasets being a prime candidate [20]. Publisher’s Note: MDPI stays neutral Protein-bound DOPA (PB-DOPA) can form via several mechanisms, including direct with regard to jurisdictional claims in incorporation of free L-DOPA in place of tyrosine into a growing polypeptide chain [21]. published maps and institutional affil- Alternatively, L-DOPA can be formed via hydroxyl attack on tyrosine residues by a reactive iations. oxygen species (ROS) [21–25], independent of free L-DOPA. Similarly, hydroxyl radical attack on the phenyl ring of phenylalanine residues forms isomers of tyrosine [22,26], the position of the hydroxyl group of these isomers (meta and ortho) differing from that of the protein amino acid L-tyrosine (para-tyrosine) (Figure1). It is known that free L-DOPA can Copyright: © 2021 by the authors. induce oxidative stress, resulting in ROS generation via interaction with iron and copper Licensee MDPI, Basel, Switzerland. to produce Fenton reaction products [27], which leads to further hydroxylation of free or This article is an open access article protein incorporated tyrosine, thereby producing free and PB-DOPA [5,22]. distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Proteomes 2021, 9, 24. https://doi.org/10.3390/proteomes9020024 https://www.mdpi.com/journal/proteomes Proteomes 2021, 9, x Proteomes 2021, 9, 24 2 of 16 2 of 15 Figure 1. Structural forms of phenylalanine, para-tyrosine, meta-tyrosine, ortho-tyrosine, and L-DOPA. Hydroxyl radical attackFigure on the phenyl 1. Structural ring of phenylalanine forms of phenylalanine, residues can form isomerspara-tyrosine, of tyrosine, meta-tyrosin the canonical forme, ortho-tyrosine, being para-tyrosine, but canand also L-DOPA. form meta-tyrosine Hydroxyl and ortho-tyrosine;radical attack hydroxyl on the attack phenyl of tyrosine ring of can phenylalanine also form L-DOPA. residues can form isomers of tyrosine, the canonical form being para-tyrosine, but can also form The misincorporation rate of canonical amino acids in general is reported to be approx- meta-tyrosine andimately ortho-tyrosine; one in 10,000; hydroxyl this low attack level of of misincorporation tyrosine can also makes form it difficult L-DOPA. to detect DOPA incorporation or other misincorporated amino acids [18,21,28,29]. However, it is possible The misincorporation rate of canonical amino acids in general is reported to be ap- that misincorporation levels of NPAAs could be higher since they can escape proofreading proximately one inmechanisms 10,000; this that low have level evolved of misincorporation to detect the mistaken makes incorporation it difficult of a to canonical detect amino DOPA incorporationacid. or As other proteomics misincorporated utilises mass spectrometers, amino acids which [18,21,28,29]. are concentration-dependent However, it is detec- possible that misincorporationtors, methodologies levels should of beNP appliedAAs could to enhance be higher the ability since to detect they these can low-abundanceescape proofreading mechanismsspecies (for that a comprehensive have evolved review, to detect see Steele the mistaken et al., 2021 [incorporation28]). The most sensitive of a ca- way to nonical amino acid.detect As low-abundanceproteomics utilises species mass in exploratory spectrometers, proteomics which is the are use concentration- of data-independent acquisition (DIA), but this requires prior identification of the peptide species and its frag- dependent detectors, methodologies should be applied to enhance the ability to detect ments for a searchable spectral library, especially in instances of modified peptides [29]. By these low-abundanceproducing species synthetic (for a comprehensive peptides for subsequent review, proteomic see Steele analysis et al., and2021 library [28]). inclusion,The it most sensitive waybecomes to detect possible low-abundance to identify very species low levels in exploratory of the modified proteomics proteoform. is the However, use the of data-independentproduction acquisition of such (DIA), a synthetic but this library requires is prohibitively prior identification expensive and of the the libraries peptide currently species and its fragmentsavailable for do nota searchable contain NPAA spectral modifications library, [ 30especially]. in instances of mod- ified peptides [29]. ByThe produc best measureing synthetic of the global peptides oxidative for state subsequent of a proteome proteomic is the level analysis of methionine oxidation, which occurs via the addition of a single oxygen atom, creating methionine and library inclusion,sulfoxide it becomes with a masspossible shift to of identify +15.99 Da, very homologous low levels to of that the of modified hydroxylation pro- [31,32]. teoform. However,However, the production protein extraction of such anda synthetic preparation library can increase is prohibitively the likelihood expensive of forming oxi- and the libraries currentlydised methionine available and do even not electrospray contain NPAA ionisation mo candifications result in this [30]. modification [29,33,34]. The best measureDue to of the the multiple global possibleoxidative end-points state of a of proteome hydroxyl attack, is the thelevel identification of methionine of oxidised oxidation, which phenylalanineoccurs via the may addition help in determining of a single if oxygen PB-DOPA atom, has been creating formed methionine from incorporation sulfoxide with a massor from shift hydroxyl of +15.99 attack. Da, Within homo plantlogous and animalto that tissues, of hydroxylation the conversion [31,32]. of tyrosine to L-DOPA can be carried out by the enzyme tyrosinase, which has also been used in indus- However, proteintrial extraction synthesis and of L-DOPA preparation and is can essential increase to the the production likelihood of dopamineof forming in theoxi- human dised methioninebrain and [33 even,34]. Unlike electrospray oxidation reactions,ionisation the enzymecan result tyrosinase in this has themodification capacity to generate [29,33,34]. Due to peptide-bondedthe multiple possible DOPA (from end-poin PB-tyrosinets of hydroxyl conversion) attack, without the generating identification other of oxidised oxidised phenylalanineamino acidmay residues help in such determining as ortho- and if meta-tyrosine.PB-DOPA has been formed from in- corporation or from hydroxylTo overcome attack. the poor Within detection plant of and low-ion-abundance animal tissues, peptides the conversion from PB-DOPA of pro- teoforms, a method for creating a positive control spectral library needs to be produced tyrosine to L-DOPA can be carried out by the enzyme tyrosinase, which has also been to enable future application to clinical samples by enabling DIA analyses. A method for used in industrialgenerating synthesis aof positive L-DOPA control and spectralis essential library to andthe