Proteomic biomarker discovery for the monogenic disease cystic fibrosis

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<ul><li><p>Review</p><p>10.1586/14789450.4.2.199 2007 Future Drugs Ltd ISSN 1478-9450 199www.future-drugs.com</p><p>Proteomic biomarker discovery for the monogenic disease cystic fibrosisDeborah Penque</p><p>Instituto Nacional de Sade Dr Ricardo Jorge, Laboratrio de Protemica, Centro de Gentica Humana, 1649-016-Lisboa, PortugalTel.: + 351 217 508 137Fax: + 351 217 526 410deborah.penque@insa.min-saude.pt</p><p>KEYWORDS: 2DE, CF-modifier gene, CFTR, CFTR-binding partner, chronic lung disease biomarker, cystic fibrosis, mass spectrometry, proteomics</p><p>Proteomics was initially viewed as a promising new scientific discipline to study complex disorders such as polygenic, infectious and environment-related diseases. However, the first attempts to understand a monogenic disease such as cystic fibrosis (CF) by proteomics-based approaches have proved quite rewarding. In CF, the impairment of a unique protein, the CF transmembrane conductance regulator, does not completely explain the complex and variable CF clinical phenotype. The great advances in our knowledge about the molecular and cellular consequences of such impairment have not been sufficient to be translated into effective treatments, and CF patients are still dying due to chronic progressive lung dysfunction. The progression of proteomics application in CF will certainly unravel new proteins that could be useful as biomarkers either to elucidate CF basic mechanisms and to better monitor the disease progression, or to promote the development of novel therapeutic strategies against CF. This review will summarize the recent technological advances in proteomics and the first results of its application to address the most important issues in the CF field.</p><p>Expert Rev. Proteomics 4(2), 199209 (2007)</p><p>The great challenge for modern medicine afterthe genome sequencing programme is the inte-grated examination of gene expression of theentire genome under physiological or patho-logical conditions, and how this expressionpattern defines and predicts the different statesof disease progression [1]. </p><p>Proteomics, the large-scale study of proteinprofiles under given times/conditions, is revo-lutionizing the way in which disease mecha-nisms are understood and how novel bio-markers and therapeutic interventions arediscovered in the postgenomic era [2]. </p><p>Initially viewed as a promising new scien-tific discipline for unraveling the complexorchestration of gene products in polygenicdiseases and the basic mechanisms in infec-tious and environmental disorders, proteomicsnow appears to be a determinant for the studyof monogenic diseases, in which the know-ledge of the functional defect of a unique geneand its product is far from being adequate forits management. </p><p>This is the case for cystic fibrosis (CF), themost common recessive monogenic disorder inCaucasians, which is caused by mutations in agene coding for a cyclic AMP (cAMP)-regu-lated chloride channel, the CF transmembraneconductance regulator (CFTR) [3]. CFTR iscritically involved in the regulation of epithelialsurface fluid composition of several organs,such as sweat glands and ducts, airways, pan-creas, intestine and the reproductive system.Absence of CFTR function leads to a multisys-temic disorder, which includes elevated sweatCl- concentrations, abnormal viscous mucusresponsible for progressive respiratory infectionand dysfunction, gastrointestinal disease, andinfertility [46]. </p><p>Since the cloning of the CFTR gene, a greatdeal of knowledge about the mutational basis ofthe disease, CFTR expression and function, andthe functional consequences of the most com-mon mutations in CFTR have been derived[46]. Despite these rapid advances in our under-standing of the molecular determinants of CF,</p><p>CONTENTS</p><p>Biological purposes</p><p>Proteomics tools</p><p>Cystic fibrosis</p><p>Proteomics-based approaches in cystic fibrosis lung disease research </p><p>Expert commentary &amp; five-year view </p><p>Key issues</p><p>References </p><p>Affiliation </p><p>For reprint orders, please contact reprints@future-drugs.com</p><p>http://www.future-drugs.com</p></li><li><p>Penque</p><p>200 Expert Rev. Proteomics 4(2), (2007)</p><p>none of the approved treatments can currently correct the bio-chemical defect, and the majority of CF patients die from achronic, progressive, lung dysfunction. </p><p>Proteomics-based approaches have the potential to provideadditional information on CF pathogenesis that could be cru-cial to the identification of new CF diagnostic/prognosticbiomarkers, as well as for the development of novel therapeuticstrategies for CF. </p><p>This review summarizes recent technological advances inproteomics and the first steps of its application to address themost important issues in the CF pulmonary field. </p><p>Biological purposesProteomics employs a variety of technologies based on highlyefficient methods of separation and analysis of proteins in orderto characterize, study and understand the proteome (set of pro-teins) of a living system at as large a scale as possible (FIGURE 1). </p><p>Proteomics has three main biological purposes:</p><p> Spatial and temporal characterization of protein expression ina cell or tissue for the identification of an entire protein setand their post-translational modifications. This approach canprovide key information about the entire protein profile (as aprotein expression map or catalog) of cells/tissues. Proteinsassociated directly or indirectly with a disease can be mappedspatially/temporally to the potential cell/tissue targets [7]. </p><p> Quantitative/qualitative comparative study of global changesin protein expression between treated and nontreated and/ornormal and diseased cells to look for toxic effects/responses ordisease diagnostic and prognostic biomarkers, respectively [8].</p><p> Functional proteome study. One example is the characteriza-tion of protein complexes to provide functional identificationof proteinprotein, DNA/RNAprotein or drugproteininteractions. Identification of such complexes and moleculesthat can regulate these interactions is of great interest sincethey may subsequently be used as targets for therapeutic drugscreening [9]. Another example is to detect functional changesin the entire proteome by using chemical probes directedtoward the active sites of specific classes of enzymes: a methodknown as activity-based protein profiling (ABPP) [10].From any of these proteomics-based biological applications,</p><p>a consolidated list of proteins, also referred to as biomarkercandidates, can be generated using any of the proteomics-based approaches described below (discovery phase; FIGURE 1).The subsequent phase is the validation of these biomarkersbefore they can be implemented as validated biomarkers forthe purposes for which they were generated (FIGURE 1). </p><p>Proteomics tools2D gel electrophoresis &amp; mass spectrometry-based proteomicsProteomics was traditionally associated with 2D gel electro-phoresis (2DE) for the separation and visualization of proteins,which was followed by protein characterization using mass spec-trometry (MS) and bioinformatics [11]. The first dimension of2DE consists of isoelectric focusing, during which the proteinsare separated on the basis of their isoelectric point in an immo-bilized pH gradient gel (IPG). This IPG is then applied on theedge of a second gel, a slab sodium dodecyl sulfate polyacryl-amide gel electrophoresis, in which the proteins undergo anadditional separation according to their molecular weight under</p><p>Figure 1. Workflow paradigm illustrating different proteomics-based approaches and major steps required for proteomic biomarker discovery, validation and implementation. ABPP: Activity-based protein profiling; ESI: Electrospray ionization; FFPE: Formalin-fixed, paraffin-embedded; ICAT: Isotope-coded affinity tag; IMAC: Immobilized metal affinity chromatography ; iTRAQ: Isobaric tags for relative and absolute quantitation; LC: Liquid chromatography; MALDI: Matrix-assisted laser desorption/ionization; MS/MS: Tandem mass spectrometry; MudPIT: Multidimensional protein identification technology; PTM: Post-translational modification; SELDI: Surface-enhanced laser desorption/ionization; SILAC: Stable isotope labeling by amino acids in cell culture; TOF: Time-of-flight.</p><p>Biologicalpurpose</p><p>Spatial and temporalcharacterization of proteinexpression (including PTMof proteins</p><p>Quantitative/qualitativecomparative study of globalchanges in protein expression(i.e., healthy disease;nontreated treated)</p><p>Functional proteome study(e.g., proteinprotein interaction and enzyme activity profiling)</p><p>Discovery phase Validation/implementation phase</p><p>Proteomics technical platform</p><p>Imaging MALDI-TOF</p><p>SELDI-TOF</p><p>Protein microarrays</p><p>2D gel2D map computer analysis</p><p>Spots</p><p>MudPIT-LC Integrated to</p><p>MALDI-TOF/TOFESI-MS/MS</p><p>Bioinformatics</p><p>Biomarker validation(immunocytochemistry, western blotting, northern blotting etc.)</p><p>Biomarker implementation</p><p>Biospecimens andsample preparation(Fresh/FFPE cells/tissues/fluids)</p><p>Total protein extracts; isolated cells from tissue by laser microdissection microscopy; prefractionated organelles; isolated protein complexes; proteins purified by depletion/fractionation/enrichment techniques (e.g., phosphorylated proteins isolated by IMAC); stable isotope-labeled proteins for proteome quantification (e.g., ICAT, iTRAQ and SILAC); labeled proteins by chemical probes for activity determination (ABPP)</p><p>Expert Review of Proteomics</p></li><li><p>Proteomic biomarker discovery for cystic fibrosis</p><p>www.future-drugs.com 201</p><p>denaturing conditions. Typically, a 1000 protein spots can besimultaneously visualized on a 2DE by using standard proce-dures of protein staining, such as Coomassie blue, colloidal blueand MS-compatible silver stain [11]. Other choices include pre-gel (fluorescence 2D differential in-gel electrophoresis) or post-run labeling with fluorescent dyes (SYPRO Ruby) or pre-runlabeling of proteins with radioisotopes (e.g., 35S-methionine or32P-phosphorous) [12]. Subsequent analysis of the spot patternon a gel by specialized software (a variety of software packagesare commercially available) enables multiple-gel comparison,whereby quantitative and qualitative changes are detected. Adatabase of 2D maps can be created and made available on awebsite. In addition to 2D images, most 2D maps containinformation about the isoelectric point and molecular weight ofthe protein spots, comparison of gels, immunoblotting resultsand MS data [13]. </p><p>The new generation of MS instrumentation has played a cru-cial role in proteomics due to its high automation and sensitiv-ity, which require femto- (10-15M) to attomolar (10-18M) con-centrations of peptide or protein material [14]. The mostpopular systems are matrix-assisted laser desorption/ionization(MALDI) time-of-flight (TOF) MS and electrospray ioniza-tion (ESI) MS [14]. The protein spot excised from the gel isdigested, usually with trypsin, and the resulting mixture of pep-tides is introduced into the MS and analyzed. Two specifictypes of protein data can be obtained:</p><p> Peptide-mass fingerprinting, which involves the determinationof the masses of all peptides in the digest;</p><p> Amino acid sequence of peptides, also called peptide-sequence tags, which are obtained by ESI tandem MS(MS/MS) or by MALDI equipped with a post-source decayapparatus or additional MS/MS (MALDI-TOF/TOF) forisolation of peptide ions and their subsequent fragmentationand microsequencing.</p><p>Peptide-mass fingerprints and peptide-sequence tags are usedto search a predicted mass map or protein sequence within adatabase to identify the protein of interest. The candidate pro-teins are ranked from a list of the most closely matched candi-dates using various scoring algorithms. If there is no match withany known sequence, new proteins and genes can be identifiedif enough sequence information is obtained. </p><p>The major limitation of current proteomics-based approachesthat combine 2DE and MS is the limited ability of 2DE toresolve low-abundance proteins and hydrophobic basic proteins[15]. A low capability to quantify proteins and peptides in complexmixtures has also been attributed to 2DE [15]. Sample prefrac-tionation or affinity-based protein purification that reduce thecomplexity of protein mixtures have increased the visualizationcapability of less abundant proteins by the 2DE system [11]. </p><p>Gel-free mass spectrometry-based proteomics In recent years, strategies to entirely circumvent the need for 2DEhave been developed [16]. One of these utilizes liquid chromato-graphy (LC) coupled directly to MS/MS. Complex mixtures of</p><p>proteins can be digested and the resulting peptides selectivelyseparated by affinity high-performance LC (according to theirhydrophobicity, charge, polarity, size or binding characteristics)and then directly analyzed by MS/MS [17]. The possibility ofeluting multiple subsets of peptides (multidimensional proteinidentification technology) allows for a dramatic increase in thetotal number of peptides that can be resolved and for whichMS/MS data can be collected [17]. Although a more comprehen-sive cataloging of proteome composition is provided, this tech-nique, per se, is not able to give reliable quantitative informationthat is crucial in disease biomarker discovery. </p><p>To overcome this limitation, new methods based on selectiveisotope peptide labeling have emerged to improve the quantita-tive comparison of control and experimental samples [16]. Stableisotope labeling by amino acids in cell culture (SILAC) isachieved in vivo via biosynthetic incorporation of stable iso-tope-containing amino acids during protein synthesis [18]. Iso-tope-coded affinity tags (ICAT), chemicals that derivatizecysteine residues, are preceded by cell lysis and protein isolation[19]. Isobaric tags for relative and absolute quantitation(iTRAQ) and O16/O18 exchange are used during or after pro-teolytic digestion, respectively [20,21]. In general, these methodsare based on reacting proteins with chemically identical formsof the above reagents that contain a linker of a light or heavytag [16]. After labeling, the samples are combined and the mix-ture analyzed by LC/MS. During the analysis, the peptidescommon to both control and experimental samples retain thesame chemical properties, and thus are detected as peak pairs,differing in mass spectral peak height or peak area, which areused to determine the relative quantification. A limitation ofthis approach is that it does not provide information aboutpost-translational modifications. For that, similar chemicalstrategies have been introduced to evaluate the post-transla-tional modification state of proteins, such as phosphorylationand glycosylation [22]. For the functional analysis of proteins, achemical approach called ABPP has been developed with amenu of chemical probes that can be used either separately orin combination to discover enzyme activities associated withdiscrete physiological and/or pathological states [23]. </p><p>Array-based proteomics Protein expression profiling by using surface-enhanced laser des-orption/ionization (SELDI) TOF is another methodology thatexpands the spectrum of tools available for proteomics...</p></li></ul>

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