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Editorial

Challenges and prospects of proteomics of

non-model organisms

The elucidation of the double helical structure of DNA, andthe mechanistic implications summarized in one sentence (Ithas not escaped our notice that the specific pairing we have

postulated immediately suggests a possible copying mechanism for

the genetic material.) at the end of the seminal articlepublished in 1953 by James D. Watson and Francis H. C.Crick[1], represented the birth of molecular biology. Less than50 years later, the first draft of the entire human genome hadbeen sequenced [2,3], heralding the new era of genomics.With an increasing list of genomes of organisms across alldomains of life being routinely sequenced, it becomes clearthat a large gap exists in predicting the phenotype from thegenotype. In other words, understanding the genome alonedoes not allow obvious extrapolations into the complexbiology that occur downstream from the gene. Proteins arethe major workhorses of biological systems, the targets ofnatural selection, and their abundance is regulated bypost-transcriptional and post-translational processes thatcannot be detected, or even inferred, only by genomic andtranscriptomic analyses. Gene regulation gives the cell controlover structure and function, and is the basis for cellulardifferentiation, morphogenesis and the versatility and adapt-ability of any organism. Gene regulation may also serve as asubstrate for evolutionary change, since control of the timing,location, and amount of gene expression can have a profoundeffect on the phenotype of a cell or multicellular organism,and phenotypic plasticity impacts on diversification andspeciation[4].

At any time point in the natural history of a living system,the genotypephenotype map is the outcome of very complexdynamics that include environmental effects. Bridging thegap between the genotype and the phenotype is synonymouswith understanding these dynamics, and this goal demands aquantitative biology approach. The need for the completepicture engendered the development of new tools (mostnotably biological mass spectrometry [5]) and even a newfield, proteomics[6,7]. Established in the 1990s, as a powerfulanalytical technique, proteomics has catalyzed an expansionof the scope of biological studies from the reductionist

approach of conventional protein chemistry to the paralleland rapid proteome-wide measurements of hundreds tothousands of proteins. The subsequent development ofquantitative MS techniques for the simultaneous study ofproteins, ultimately the whole proteome, that are expressedin response to changes in gene activity, has represented amajor advance in the field over the first decade of the XXIcentury[8].

Although significant advances in the comprehensiveprofiling of proteins have occurred in model organisms (i.e.those that, because of their small size and short generationtimes, facilitate experimental laboratory research), proteo-mics research in non-model organisms, albeit representingthe vast majority of the biodiversity that exists on Earth[9],has not advanced at the same pace. There are several reasonsthat hamper the application of high-throughput proteomicsto non-model, particularly to non-genome-sequenced, organ-isms. On the one hand, peptide sequencing via MS/MSrequires a great deal ofde novointerpretation of product ionmass spectra [10], involving the manual intervention of anexpert mass spectrometrist, to subsequently carryoutcross-species, homology-based database searches[11]. Auto-matedde novo peptide sequencing remains a challenge, andeven a simple MS/MS spectrum may require minutes for atrained expert to interpret. On the other hand, the applicationof proteomic approaches to non-model organisms whosegenome is not available or ill-defined entails the principalchallenge of making it necessary to infer protein functionalitythrough the evolutionary conservation of gene sequences andprotein domains between species. In addition, shotgunprotocols do not work for non-genome-sequenced organisms,precluding the use of shotgun and targeted proteomics for therelative or absolute quantification of proteins. Notwithstand-ing these limitations, the proteomics of non-model organismsalso presents its own strengths. Hence, proteogenomics[12]and proteotranscriptomics[13] offer the possibility of refining,even proof-checking, species-specific genome and tran-scriptome databases using proteomics data. Further opportu-nities of proteomics investigations of non-model organisms

A v a i l a b l e o n l i n e a t w w w . s c i e n c e d i r e c t . c o m

ScienceDirect

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J O U R N A L O F P R O T E O M I C S 1 0 5 ( 2 0 1 4 ) 1 4

http://dx.doi.org/10.1016/j.jprot.2014.04.0341874-3919/ 2014 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jprot.2014.04.034http://dx.doi.org/10.1016/j.jprot.2014.04.034http://dx.doi.org/10.1016/j.jprot.2014.04.034http://dx.doi.org/10.1016/j.jprot.2014.04.034http://dx.doi.org/10.1016/j.jprot.2014.04.034http://dx.doi.org/10.1016/j.jprot.2014.04.034http://localhost/var/www/apps/conversion/tmp/scratch_1/Unlabelled%20imagehttp://localhost/var/www/apps/conversion/tmp/scratch_1/Unlabelled%20imagehttp://crossmark.crossref.org/dialog/?doi=10.1016/j.jprot.2014.04.034&domain=pdf

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variation in proteins versus gene regulatory changes as thebasis for adaptive variation in phenotype. The importance ofgene regulation in natural populations has been inferred frommeasurements of variation in gene expression using a varietyof techniques. The most widely-used approach has been tomeasure variation in mRNA levels for specific genes usingmicroarrays developed for model organisms [21]. While this

approach is powerful, allowing the simultaneous assessmentof transcript variation in many genes, it makes the assump-tion that there is a close link between transcript abundanceand protein levels. Further, it requires the existence of amicroarray with homologous loci. For non-model organismswhere such resources don't exist, an alternative is to directlymeasure variation in the amounts of specific proteins asquantified using recently developed proteomics techniques.Fortunately, deconstructing complex venom phenotypes isnow within the reach of proteomic technologies [13], andunderstanding evolutionary trends across venoms, and theirwithin- and between-species toxicological and immunologi-cal divergences and similarities, is of applied importance for

generating broad-ranging polyspecific antivenoms withwhich to fight the neglected pathology of snakebiteenvenoming[2224].

Theapplication of the powerfulCombinatorialPeptideLigandLibrary (CPLL) technique for thein-depth exploration of the traceproteome of Champagne wines, and the proteomics-basedidentification of the composition and manufacturing recipe of a2500-year old sourdough bread unearthed by archeologicalexcavations in a Subeixi cemetery in China, complete the

journey across a small region of the vast landscape ofnon-model systems amenable to proteomics studies.

The community working on model organisms is growingsteadily and the number of model organisms for whichproteome data are being generated is continuously increasing.A few years ago, the Journal of Proteomics devoted a SpecialIssue to[25]. More recently, a Human Proteome Organisation(HUPO) initiative on model organism proteomes (iMOP,http://www.imop.uzh.ch) was approved at the HUPO Ninth AnnualWorld Congress in Sydney, 2010 [26]. The iMOP initiative seeksto promote proteomics within the model organism commu-nities working on all non-humanspecies, to integrate and linkproteome and other organism-specific databases. Increasedinteractions among model organism proteomics researchersare also expected to lead to the development of jointresources and standards that should ultimately lead toimproved and innovative science.

Model organisms represent an important testing ground forthe development and validation of analytical approaches forproteomics. The research community on non-model organismsmayalso benefit fromthese advances, particularlyif performedon close relatives with well annotatedreference sequences.Thepromise of large-scale whole-genome sequencing projects suchas the Genome 10K Project (https://genome10k.soe.ucsc.edu), aproposal to obtain whole-genome sequences for 10,000 repre-sentative vertebrate species spanning evolutionary diversityacross living mammals, birds, nonavian reptiles, amphibians,and fishes[27], aims to anchor investigation of relatives in adiversity of phylogenetic neighborhoods. However, researchersof non-model organisms must also be well aware that, ashighlighted in the articles of this Special Issue, proteomics in

their non-model organisms of interest, although challenging, ispossible given sufficient human resources and commitment.Thetime andefforts invested in arranging thisSpecial Issue willhave been worthwhile if its reading attracts new explorers todelve into the largely unexplored field of proteomics ofnon-model organisms.

R E F E R E N C E S

[1] Watson JD, Crick FHC. A structure for deoxyribose nucleicacid. Nature 1953;171:7378.

[2] International Human Genome Sequencing Consortium.Initial sequencing and analysis of the human genome.Nature 2001;409:860921.

[3] Venter JC, et al. The sequence of the human genome. Science2001;291:130451.

[4] Pfennig DW, Wund MA, Snell-Rood EC, Cruickshank T,Schlichting CD, Moczek AP. Phenotypic plasticity's impactson diversification and speciation. Trends Ecol Evol

2010;25:45967.[5] Calvete JJ. The expanding universe of mass analyzer

configurations for biological analysis. Methods Mol Biol2014;1072:6181.

[6] KloseJ. From 2-D electrophoresis to proteomics. Electrophoresis2009;30:S1429.

[7] James P. Protein identification in the post-genome era: therapid rise of proteomics. Q Rev Biophys 1997;30:279331.

[8] Eyers CE, Gaskell S, editors. Quantitative proteomics. RSCPublishing. ISBN 978-1-84973-808-8; 2014. p. 1371.

[9] Hedges SB. The origin and evolution of model organisms. NatRev Genet 2002;3:83849.

[10] Ma B, Johnson R. De novo sequencing and homologysearching. Mol Cell Proteomics 2012;11 [O111.014902].

[11] Junqueira M, Spirin V, Balbuena TS, Thomas H, Adzhubei I,

Sunyaev S, et al. Protein identification pipeline for thehomology-driven proteomics. J Proteomics 2008;71:34656.

[12] Armengaud J, Trapp J, Pible O, Geffard O, Chaumot A,Hartmann EM. Non-model organisms, a species endangeredby proteogenomics. J Proteomics 2014;105:518.

[13] Calvete JJ. Next-generation snake venomics: protein-locusresolution through venom decomplexation. Expert RevProteomics Mar. 29 2014 [Epub ahead of print].

[14] Deisenhofer J, Epp O, Miki K, Huber R, Michel H. Structure ofthe protein subunits in the photosynthetic reaction centre ofRhodospeudomonas viridisat 3 resolution. Nature1985;318:61824.

[15] http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1962/kendrew-lecture.pdf.

[16] Ellegren H. Genome sequencing and population genomics innon-model organisms. Trends Ecol Evol 2014;29:5163.

[17] Durban J, Prez A, Sanz L, Gmez A, Bonilla F, Rodrguez S,et al. Integrated omicsprofiling indicates that miRNAs aremodulators of the ontogenetic venom composition shift inthe Central American rattlesnake,Crotalus simus simus. BMCGenomics 2013;14:234.

[18] Gibbs HL, Sanz L, Calvete JJ. Snake population venomics:proteomics-based analyses of individual variation revealssignificant gene regulation effects on venom proteinexpression in Sistrurus rattlesnakes. J Mol Evol 2009;68:11325.

[19] Diz AP, Martnez-Fernndez M, Roln-Alvarez E. Proteomicsin evolutionary ecology: linking the genotype with thephenotype. Mol Ecol 2012;21:106080.

[20] Tomanek L. Introduction to the symposium ComparativeProteomics of Environmental and Pollution Stress. IntegrComp Biol 2012;52:6225.

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[21] Whitehead A, Crawford DL. Variation within and amongspecies in gene expression: raw material for evolution. MolEcol 2006;15:1197211.

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[23] Gutirrez JM, Solano G, Pla D, Herrera M, Segura , Villalta M,

et al. Assessing the preclinical efficacy of antivenoms: fromthe lethality neutralization assay to antivenomics. Toxicon2013;69:16879.

[24] Williams DJ, Gutirrez JM, Calvete JJ, Wster W,Ratanabanangkoon K, Paiva O, et al. Ending the drought: newstrategies for improving the flow of affordable, effectiveantivenoms in Asia and Africa. J Proteomics 2011;74:173567.

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[27] Genome 10K Community of Scientists. A proposal to obtainwhole-genome sequence for 10,000 vertebrate species. JHered 2009;100:65974.

Juan J. CalveteInstituto de Biomedicina de Valencia CSIC, Jaime Roig 11,

46010 Valencia, Spain

Tel.: +34 963391778.E-mail address:jcalvete@ibv.csic.es.

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