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Advances in the Development of Human Protein Microarrays Expert Review of Proteomics ISSN: 1478-9450 (Print) 1744-8387 (Online) Journal homepage: http://www.tandfonline.com/loi/ieru20 Advances in the development of human protein microarrays Jessica G. Duarte & Jonathan M. Blackburn To cite this article: Jessica G. Duarte & Jonathan M. Blackburn (2017) Advances in the development of human protein microarrays, Expert Review of Proteomics, 14:7, 627-641, DOI: 10.1080/14789450.2017.1347042 To link to this article: http://dx.doi.org/10.1080/14789450.2017.1347042 Accepted author version posted online: 23 Jun 2017. Published online: 04 Jul 2017. Submit your article to this journal Article views: 22 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ieru20 Download by: [202.152.71.55] Date: 09 July 2017, At: 07:36 EXPERT REVIEW OF PROTEOMICS, 2017 VOL. 14, NO. 7, 627–641 https://doi.org/10.1080/14789450.2017.1347042 REVIEW Advances in the development of human protein microarrays Jessica G. Duartea and Jonathan M. Blackburn b aCancer Immunobiology Laboratory, Olivia Newton-John Cancer Research Institute/School of Cancer Medicine, La Trobe University, Heidelberg, Australia; bInstitute of Infectious Disease and Molecular Medicine & Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Observatory, South Africa ABSTRACT ARTICLE HISTORY Introduction: High-content protein microarrays in principle enable the functional interrogation of the Received 20 February 2017 human proteome in a broad range of applications, including biomarker discovery, profiling of immune Accepted 22 June 2017 responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein KEYWORDS and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are Protein microarrays; under active technological development and a range of different protein microarrays are now commer- proteomics; biomarker cially available. However, deciphering the differences between these platforms to identify the most discovery; antibody profiling suitable protein microarray for the specific research question is not always straightforward. Areas covered: This review provides an overview of the technological basis, applications and limita- tions of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays. Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling appli- cations, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality. 1. Introduction gene expression, copy number variation, splice variation, poly- morphic variation, DNA methylation, and transcription factor Since the introduction of DNA microarrays in the mid-1990s, a binding sites. wide range of analogous microarray technologies have been In the proteomics field, microarray technologies today spawned that enable a multitude of applications across the include full-length protein arrays, protein fragment arrays, biological and biomedical sciences and which encompass peptide arrays, antibody arrays, reverse-phase arrays and tis- genomic, transcriptomic, proteomic, glycomic, epigenomic, sue arrays. In this Review, we provide an overview of recent and drug discovery research areas, among others. At their technological advances in the development of protein micro- simplest, microarrays can be considered as lab-on-chip tools arrays, with a specific focus on full-length protein and protein consisting of multiple different biomolecules that are immo- fragment microarrays, discussing the technological basis, bilized at spatially addressable locations on a surface and applications and limitations of some of the most commonly which can function as discrete probes in downstream, highly used protein microarray platforms. Technological advances in miniaturized, multiplexed assays. The parallel interaction of the antibody microarray field have been reviewed recently such immobilized probes with analytes contained in complex elsewhere [3–7] so are not dealt with again here. sample solutions results in the generation of vast amounts of Early research in the protein microarray field focused initi- quantitative, functional and interaction-based information for ally on simple technical demonstrations that instrumentation analysis [1,2]. and surfaces adapted directly from the DNA microarray field The underlying microarray technologies themselves remain could also be used to fabricate protein microarrays: commer- under active development in several areas, including diversifi- cially-available arrayers were used to print nanolitre volumes cation of content, improvement in surfaces and broadening of of different purified proteins onto chemically derivatized glass application areas. For example, in the genomic field, DNA slides, resulting in high density, spatially defined patterns; microarray technology has been revolutionized over the discrete locations on those prototype microarrays were iden- years by dramatically expanding the number of probes on tified through on-chip protein interaction assays, using fluor- the arrays while concomitantly reducing the feature sizes, escently labeled, known binding partners; and assays were improving the underlying surfaces, and developing many read out using DNA microarray scanners [8]. From those new application areas, including inter alia global analysis of CONTACT Jonathan M. Blackburn [email protected] Institute of Infectious Disease and Molecular Medicine & Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa © 2017 Informa UK Limited, trading as Taylor & Francis Group 628 J. G. DUARTE AND J. M. BLACKBURN relatively modest beginnings, protein microarrays have devel- throughput purification strategies. Insect cell expression sys- oped progressively into valuable proteomic tools that today tems have therefore found favor with several groups since, can contain tens of thousands of different proteins, including once transfected with a suitable baculoviral vector, expres- entire proteomes in some cases, and which are capable of sion of many different eukaryotic proteins with more com- addressing numerous different biological questions, with plex PTMs can be achieved in parallel in a few days, albeit potential applications in biomarker discovery as well as in with typically lower yields, and downstream lysis methods the quantitative analyses of protein function and interactions. are much milder than for yeast [10]. By contrast, mammalian However, realization of the true potential of modern protein expression systems are less simple to establish and maintain microarray technologies relies on the effective integration and in high throughput, while cell-free transcription-translation application of various components of the overall system, systems can be good sources of polypeptide, but often including high throughput protein expression and purification, require optimization for expression of individual folded, protein immobilization, assay development, signal detection, functional proteins. data processing and data analysis [9]. Classic limiting factors to Following expression, high throughput, parallel purification the wider uptake of protein microarrays by the community of recombinant proteins to near-homogeneity is also not have included the availability of protein content in a form always straightforward, since in many cases, short peptide suitable for arraying, the development of surface chemistries tags (e.g. the hexa-His tag) can be occluded, necessitating to preserve the folded structure and function of proteins on purification under denaturing conditions [11]. The protein immobilization, and the availability of assays for all the arrayed microarray field has thus in general gravitated now toward proteins; these factors are discussed briefly in turn below. use of larger protein domains as affinity tags (e.g. the glu- tathione S-transferase (GST) tag), as well as to the use of automated purification systems. Some groups, though, have adopted alternative purification strategies, combining specific 1.1. Content generation protein affinity tags with specialized array surfaces to enable One of the early decisions to be made in the protein micro- single step, in situ protein purification and surface immobiliza- array field is how the content will be generated. One obvious tion [12–17], thus circumventing the need for laborious high approach is to separate many different proteins directly from throughput pre-purification of proteins prior to array native sources, which at first sight has the advantage that the fabrication. native proteins come complete with relevant post-transla- tional modifications and potentially also with interacting part- ners. However, purification of a single native protein or 1.2. Array fabrication complex from a biological sample can be a lengthy, highly optimized
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