proteomes Review Misincorporation Proteomics Technologies: A Review Joel R. Steele 1,2 , Carly J. Italiano 2 , Connor R. Phillips 2, Jake P. Violi 1,2, Lisa Pu 2 , 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] (J.R.S.); [email protected] (J.P.V.) 2 Neurotoxin Research Group, School of Life Sciences, The University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] (C.J.I.); [email protected] (C.R.P.); [email protected] (L.P.); [email protected] (K.J.R.) * Correspondence: [email protected] Abstract: Proteinopathies are diseases caused by factors that affect proteoform conformation. As such, a prevalent hypothesis is that the misincorporation of noncanonical amino acids into a pro- teoform results in detrimental structures. However, this hypothesis is missing proteomic evidence, specifically the detection of a noncanonical amino acid in a peptide sequence. This review aims to outline the current state of technology that can be used to investigate mistranslations and misincor- porations whilst framing the pursuit as Misincorporation Proteomics (MiP). The current availability of technologies explored herein is mass spectrometry, sample enrichment/preparation, data analysis techniques, and the hyphenation of approaches. While many of these technologies show potential, our review reveals a need for further development and refinement of approaches is still required. Keywords: misincorporation; non protein amino acids; post translational modifications Citation: Steele, J.R.; Italiano, C.J.; 1. Introduction Phillips, C.R.; Violi, J.P.; Pu, L.; The “central dogma” of molecular biology suggests that the translation of one gene Rodgers, K.J.; Padula, M.P. results in the expression of a single protein [1]. However, translated proteins are known to Misincorporation Proteomics exist as multiple biological variants or proteoforms [2]. These proteoforms are the result of Technologies: A Review. Proteomes modifications to the polypeptide chain, including the addition, subtraction, or alteration 2021 9 , , 2. https://doi.org/10.3390/ of chemical groups. Such modifications can endow proteoforms with biological activity proteomes9010002 or an altered function varying from the original proteoform [3]. Any modification that occurs to a proteoform once already translated is termed a post-translational modification Received: 16 December 2020 (PTM), generating a new and distinct proteoform, adding the advantage of complexity to Accepted: 18 January 2021 Published: 21 January 2021 the proteome [4–6]. Additionally, and importantly for this review, variant proteoforms may be generated Publisher’s Note: MDPI stays neutral by mechanisms other than post-translational modification. During protein translation, with regard to jurisdictional claims in an incorrect amino acid may be inserted into the growing peptide chain, resulting in a published maps and institutional affil- modification in the final proteoform. Such errors generate new, non-native proteoforms iations. that have the potential to cause harm to the cell [7,8]. The mistranslational error rate reported in Escherichia coli (E. coli) is between 0.5–5% at any amino acid position [8]. This occurrence of errors in translation during synthesis can generate a new proteoform in the same way that cleavage or PTM can e.g., N-terminal methionine excision [8–10]. However, instead of providing an advantage to the cell, proteoforms produced through mistranslation Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. often present a burden, as they are unpredictably generated with the resulting non-native This article is an open access article proteoforms prone to misfolding [11,12]. To overcome this, the cell has sophisticated distributed under the terms and machinery to identify and degrade these proteoforms [13]. conditions of the Creative Commons The “misincorporation” of incorrect amino acids into a proteoform need not be limited Attribution (CC BY) license (https:// to the 20 canonical amino acids used in proteoform synthesis. Thousands of synthetic and creativecommons.org/licenses/by/ naturally occurring nonprotein amino acids (NPAAs) exist, also referred to as “nonpro- 4.0/). teogenic”, “noncanonical”, “noncoded”, or “non-natural” [14]. The infiltration of NPAAs Proteomes 2021, 9, 2. https://doi.org/10.3390/proteomes9010002 https://www.mdpi.com/journal/proteomes Proteomes 2021, 9, x FOR PEER REVIEW 2 of 20 Proteomes 2021, 9, 2 2 of 20 “nonproteogenic”, “noncanonical”, “noncoded”, or “non-natural” [14]. The infiltration of NPAAs into the protein translation process adds further opportunity for mistakes to be madeinto the during protein translation, translation resulting process in addsdistinct further and unintentionally opportunity for produced mistakes proteoforms. to be made Suchduring misincorporation translation, resulting can cause in distinct aberrant and modifications unintentionally to proteoform produced proteoforms. structure, with Such bi- ologicalmisincorporation ramifications can causefor the aberrant cell or organism modifications that can to proteoform perturb normal structure, cellular with function. biological It ramifications for the cell or organism that can perturb normal cellular function. It has has been shown that misincorporation of an NPAA can alter the 3D structure of a pro- been shown that misincorporation of an NPAA can alter the 3D structure of a proteoform, teoform, resulting in aggregation [15]. Such misfolding and aggregation of proteoforms is resulting in aggregation [15]. Such misfolding and aggregation of proteoforms is known known to be a hallmark of numerous degenerative neurological diseases [16] and for some to be a hallmark of numerous degenerative neurological diseases [16] and for some dis- diseases, an association already exists between exposure to NPAAs and disease develop- eases, an association already exists between exposure to NPAAs and disease development. ment. This includes motor neuron disease (MND) [17,18], multiple sclerosis [19,20] and This includes motor neuron disease (MND) [17,18], multiple sclerosis [19,20] and neuro- neurolathyrism [21,22]. As such, the exploration of NPAA misincorporation into pro- lathyrism [21,22]. As such, the exploration of NPAA misincorporation into proteoforms to teoforms to date has largely focused on their underlying potential to trigger proteinopa- date has largely focused on their underlying potential to trigger proteinopathies and cause thies and cause neurodegeneration. To describe the study of NPAA misincorporation into neurodegeneration. To describe the study of NPAA misincorporation into the proteome, we the proteome, we introduce the term Misincorporation Proteomics (MiP), whereby during introduce the term Misincorporation Proteomics (MiP), whereby during protein translation, proteina genetically translation, encoded a genetically canonical aminoencoded acid canonical is replaced amino by a acid NPAA. is replaced by a NPAA. 2. Amino Acid Misincorporation Protein translation translation involves involves the the cognate cognate amino amino acid acid (AA) (AA) being being charged charged by its by appro- its ap- propriatepriate aminoacyl aminoacyl tRNA tRNA synthetase synthetase (aaRS; (aaRS; Figure Figure 1). 1The). The charging charging is enabled is enabled by bythe thehy- drolysishydrolysis of ofadenosine adenosine triphosphate triphosphate (ATP) (ATP) and and allows allows the formationformation ofof an an AA/aaRS/AMP AA/aaRS/AMP complex. The cognate transfertransfer RNARNA (tRNA)(tRNA) thenthen bindsbinds to to this this aaRS/AA/AMP aaRS/AA/AMP complex and an aminoacyl ester bond forms, transferringtransferring the aminoacyl group to the tRNA and releasing AMP [[23,24].23,24]. Figure 1. TheThe transfer transfer of ofamino amino acids acids (AA) (AA) onto onto tRNA tRNA through through tRNA tRNA synthetase synthetase (aaRS) (aaRS) in the in pres- the presenceence of ATP. of ATP. ATP ATP is converted is converted to AMP to AMP activating activating thethe aaRS aaRS with with the the AA, AA, an antRNA tRNA is then is then bound bound to theto the AA AA of ofthe the AA/aaRS/AMP AA/aaRS/AMP complex complex subseque subsequentlyntly the thenow now charged charged tRNA tRNA dissociates. dissociates. Correct binding of amino acid to its aaRS is dependent on the fit between the amino acid and enzyme binding pocket [25]. If an incorrect amino acid binds the aaRS, this can be removed by a series of proofreading functions known as pre- and post-transfer Proteomes 2021, 9, x FOR PEER REVIEW 3 of 20 Proteomes 2021, 9, 2 3 of 20 Correct binding of amino acid to its aaRS is dependent on the fit between the amino acid and enzyme binding pocket [25]. If an incorrect amino acid binds the aaRS, this can be removed by a series of proofreading functions known as pre- and post-transfer editing (Figureediting (Figure2). Pre-transfer2). Pre-transfer editing editing occurs occurs before before tRNA tRNA binds binds to the to charged the charged amino amino acid. acid. Re- movalRemoval of the of the charged charged amino amino acid acid has has been been suggested suggested to to occur occur via via various various mechanisms, mechanisms, includingincluding selective selective release release by by the the aaRS aaRS enzyme, enzyme, translocation translocation to a to separate a separate hydrolytic hydrolytic ed-