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Review Proteomes Are of Proteoforms proteomes Review Review ProteomesProteomes AreAre ofof Proteoforms:Proteoforms: EmbracingEmbracing thethe ComplexityComplexity Katrina Carbonara, Martin Andonovski and Jens R. Coorssen * Katrina Carbonara, Martin Andonovski and Jens R. Coorssen * Faculties of Applied Health Sciences and Mathematics & Science, Departments of Health Sciences and FacultiesBiological of Sciences, Applied HealthBrock University, Sciences and 1812 Mathematics Sir Isaac Brock & Science, Way, St. Departments Catharines, of ON Health L2S Sciences3A1, Canada; and Biological Sciences, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON L2S 3A1, Canada; [email protected] (K.C.); [email protected] (M.A.) [email protected] (K.C.); [email protected] (M.A.) * Correspondence: [email protected] * Correspondence: [email protected] Abstract: Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplify- Abstract: Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplifying ing their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. While having a catalogue of amino acid sequences provides invaluable information, this is the Pro- While having a catalogue of amino acid sequences provides invaluable information, this is the teome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we must Proteome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we assess the myriad of proteoforms that arise at any point before, after, and between translation and must assess the myriad of proteoforms that arise at any point before, after, and between translation transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as well as and transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as newly defined species. There are numerous analytical methods currently used to address proteome well as newly defined species. There are numerous analytical methods currently used to address depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We thus dis- proteome depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We cuss both pros and cons of available approaches and where improvements or refinements are thus discuss both pros and cons of available approaches and where improvements or refinements are needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest that advances lie in transdisciplinarity via integration of current proteomic methods to yield a uni- that advances lie in transdisciplinarity via integration of current proteomic methods to yield a unified fied discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolution- discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolutionary) ary) shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptom- shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptomics. ics. We must embrace the complexity. Yes, these are the hard questions, and this will not be We must embrace the complexity. Yes, these are the hard questions, and this will not be easy ... but easy…but where is the fun in easy? where is the fun in easy? Citation: Carbonara, K.; Keywords: proteomics; top-down; bottom-up; immunoassay; mass spectrometry; two-dimensional Keywords: proteomics; top-down; bottom-up; immunoassay; mass spectrometry; two-dimensional Andonovski,Citation: Carbonara, M.; Coorssen, K.; J.R. gel electrophoresis; Western blotting ProteomesAndonovski, Are M.; of Proteoforms:Coorssen, J.R. gel electrophoresis; Western blotting EmbracingProteomes theAre Complexity. of Proteoforms:Proteomes 2021Embracing, 9, 38. https://doi.org/10.3390/the Complexity. proteomes9030038Proteomes 2021, 9, x. https://doi.org/10.3390/xxxxx Academic Editor: Julian Whitelegge Academic Editor: Julian Whitelegge Received: 1 August 2021 Accepted:Received: 291 August August 2021 2021 Published:Accepted: 3129 AugustAugust 20212021 Published: 31 August 2021 Publisher’s Note: MDPI stays neutral withPublisher’s regard to Note: jurisdictional MDPI stays claims neu- in publishedtral with maps regard and institutionalto jurisdictional affil- iations.claims in published maps and institu- (NON(NON SEQUITUR © © Wiley Wiley Ink, Ink, Inc. Inc. Dist.Dist. By ANDREWS By ANDREWS MCMEEL MCMEEL SYNDICATION. SYNDICATION. Reprinted tional affiliations. Reprintedwith permission. with permission.All rights reserved.). All rights reserved.) 1.1. IntroductionIntroduction Copyright: © 2021 by the authors. Throughout a lifespan, the genome remains essentially unaltered in every somatic cell Copyright: © 2021 by the authors. Li- Throughout a lifespan, the genome remains essentially unaltered in every somatic Licensee MDPI, Basel, Switzerland. censee MDPI, Basel, Switzerland. ofcell an of organism, an organism, the exception the exception being being epigenetic epigenetic influences influences on gene on expression, gene expression, and hence and This article is an open access article This article is an open access article proteins.hence proteins. In contrast, In contrast, the proteome the proteome is constantly is constantly changing changing and responding and responding to stimuli, to bothstim- distributed under the terms and distributed under the terms and con- internaluli, both and internal external and [external1]. The proteome [1]. The pr wasoteome first was defined first as,defined “the PROTEinas, “the PROTEin complement com- conditions of the Creative Commons ditions of the Creative Commons At- expressedplement expressed by a genOME” by a genOME” [2] but here [2] but we here will emphasizewe will emphasize how and how why and it iswhy much it is moremuch Attribution (CC BY) license (https:// tribution (CC BY) license (http://crea- complexmore complex than this than original this original definition definition implied. implied. It is this It complexityis this complexity that must that be must respected be re- creativecommons.org/licenses/by/ tivecommons.org/licenses/by/4.0/). and addressed to ensure the best possible analyses of proteomes. 4.0/). spected and addressed to ensure the best possible analyses of proteomes. Proteomes 2021, 9, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/proteomes Proteomes 2021, 9, 38. https://doi.org/10.3390/proteomes9030038 https://www.mdpi.com/journal/proteomes Proteomes 2021, 9, 38 2 of 38 Proteomes 2021, 9, x FOR PEER REVIEW 2 of 39 For the field of proteomics to effectively move forward, there must first be a discussion For the field of proteomics to effectively move forward, there must first be a discus- and broader consensus on what a ‘protein’ is. To what exactly does the word ‘protein’ sion and broader consensus on what a ‘protein’ is. To what exactly does the word ‘protein’ refer? It is refer?well known It is well that known proteins that serv proteinse as antibodies, serve as enzymes, antibodies, messengers, enzymes, and messengers, so and so much moremuch [3]. However, more [3]. the However, central dogma the central that dogmawe are so that familiar we are with so familiaris as follows: with is as follows: DNA is transcribedDNA is transcribed into mRNA into and mRNAthen translated and then into translated an amino into acid an sequence, amino acid which sequence, which is the primaryis the structure primary of structurea protein [4–6]. of a proteinAmino acids [4–6]. are Amino thus the acids backbone are thus of proteins, the backbone of pro- but they doteins, not but fully they or doeffectively not fully define or effectively its function, define localization, its function, or localization, interactions. or interactions. DNA/RNADNA/RNA coding sequence, coding mutations, sequence, and mutations, variability and of variabilitytranslational of translationalstart site are key start site are key elements thatelements reveal thatinformation reveal information about primary about protein primary structure protein and structure chemistry. and These chemistry. These elements—alongelements—along with the immense with the number immense of possible number modifications of possible modifications that can occur that at can occur at any given anypoint given before point translation, before after translation, transcription, after transcription,or in-between (e.g., or in-between RNA splicing, (e.g., RNA splic- alternativeing, splicing, alternative alternate splicing, open reading alternate frames open (AltORF) reading frames[7–9], single (AltORF) nucleotide [7–9], pol- single nucleotide ymorphismspolymorphisms (SNP) [10,11], and (SNP) mRNA [10,11 editing], and [12–17]), mRNA editingpost-translational [12–17]), post-translational modifications modifica- (PTM), andtions adducts)—each (PTM), and yield adducts)—each multiple diff yielderent multiple protein species different of proteina given speciesamino acid of a given amino sequence, andacid each sequence, such species, and each or proteoform such species,, differs or proteoform, in its biological differs function in its (Figure biological function 1) [5,18,19].(Figure Being1 the)[ 5 critical,18,19]. players Being thein the critical majority
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