New capture molecules (probes) for protein chips

This review book, without pretending of being exhaustive, will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications. It has been structured in three main chapters that treat the single basic themes of applications, types and realization of protein chips, to face the matter with a multidisciplinary approach involving: biology for capture molecules, chemistry for methods of immobilization, material engineering for support fabrication, physics for detection systems. In recent years, protein microarrays have become one of the most invaluable research tools in the field of large-scale and high-throughput biology, and their use in basic research, diagnostics and drug discovery has emerged as a great promise of medicine. The fast growth of protein chip technology is fuelled by the continuous growth of genomic information; the integration of large datasets from different approaches will result in the generation of a huge network and deepen our understanding of the molecular mechanisms of life. The actual impact of the new technology on proteomic and medical research are yet to be fully realized, opening a new era of collection and analysis information. During the last few decades, scientists have extended their investigations beyond molecular level into the field of the so-called “supramolecular chemistry”. Basic themes such as molecular self-assembly, folding, molecular recognition, host-guest chemistry, and nanoscience are often associated with this area of research. Broadly speaking, supramolecular chemistry is the study of interactions between, rather than within, molecules. While traditional chemistry focuses on strong association forces such as covalent and ionic bonds (used to assemble atoms into discrete molecules), supramolecular chemistry examines the weaker and reversible noncovalent interactions between molecules (used to organize and hold together supramolecular assemblies), including hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, hydrophilic-hydrophobic interactions and electrostatic effects. In nature, organization on the nanometer scale is crucial for the remarkable properties and functional capabilities of biological systems; thus, the use of these principles led to an increasing understanding of many biological processes based on protein structure and function. Concomitantly, nanotechnologists began to be able to take these concepts and apply them to synthetic systems based on noncovalent interactions. The development of selective “host-guest” complexes in particular, in which a host molecule recognizes and selectively binds a certain guest, can be cited as an important contribution to protein chip technology. In addition, the need for improved miniaturization and device performance in the microelectronics industry has inspired many investigations into supramolecular chemistry. It is conceivable that “bottom up” materials fabrication approaches based on supramolecular chemistry will provide a solution to the anticipated size limitations of “top down” approaches, such as photolithography, thereby providing the means to fabricate ultrasmall electronic components. Hybrid devices integrate the outstanding electronic and photonic properties of nonmolecular hard materials such as silicon to the excellent bio-recognition, chemical sensing, and related properties of designed, soft molecular materials to yield structures capable of performing disease diagnosis, environmental monitoring, controlled drug delivery, and so on. Miniaturized and parallel assay systems, in particular microarray-based analyses, allow fast, easy and parallel detection of thousands of addressable elements in a single experiment. They are applied to analyse antibody-antigen, protein-protein, protein-nucleic acid, protein-lipid and protein-small molecule as well as enzyme-substrate interactions. New trends in protein chip technology include surface chemistry, capture molecules, labeling and detection methods, high-throughput protein production, and applications to analyse entire proteomes. The challenges are to elucidate the basic cellular events mediating complex processes and those causing diseases. It is well recognised that the complexity of the proteome far exceeds that of the genome. When variables such as alternative gene splicing events, post-translational modifications and individual coding variants are taken into account, the number of different molecular protein species is likely to be at least 1-2 orders of magnitude greater than the number of genes. Conventional proteome analysis by 2D gel electrophoresis and mass spectrometry, while highly effective, has limitations and in particular may miss many proteins of interest when expressed at low abundance and is unsuited to diagnostic applications. Since the little abundant proteins are often those of the greatest diagnostic interest (e.g. cytokines and biomarkers in plasma), there is therefore an acknowledged need for other highly sensitive, specific and accessible high throughput technologies for protein detection, quantization and differential expression analysis in health and disease. For this reason, protein arrays are poised to become a central technology at the research and biotechnology levels. Protein arrays generally fall into three types. In protein function arrays, a large set of purified proteins or peptides or even an entire proteome is spotted and immobilized, then the array is used for parallel screening of a range of biochemical interactions. Protein function arrays are generally aimed at discovering protein function in fundamental research and can be used to study the effect of substrates or inhibitors on enzyme activities, protein-drug or hormone-effector interactions, or epitope mapping. In protein detection microarrays (usually referred as analytical microarrays), an array of affinity reagents (antigens or antibodies, but also peptides or aptamers), rather than the native proteins themselves, is immobilized on a support and used to determine protein abundances in a complex matrix such as plasma/serum or tissue extracts. Analytical arrays can be used to assay antibodies (for diagnosis of allergy or autoimmunity diseases) or to monitor protein expression on a large scale. In a third category of protein arrays (usually referred as reverse phase microarrays), tissues, cell lysates, or serum samples are spotted on the surface and probed with one antibody per analyte for a multiplex readout. For construction of arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high throughput production. Recent advances involve the use of whole phage as selective and specific probes, possessing distinct advantages as durability, stability, standardization and low-cost production. For capture arrays and protein function analysis, it is clearly important that proteins should be correctly folded and functional. On the other hand, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying auto-antibodies and selecting ligand binding proteins, where linear epitopes are recognised. The immobilisation method used should be reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity. Both covalent and noncovalent methods of protein immobilisation are used with various pros and cons. What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labelling is used for comparison of different samples with different fluorophores. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. On the other hand, label-free detection methods, including Mass Spectrometry and Surface Plasmon Resonance, avoid alteration of ligand. The design of capture arrays, particularly when exposed to heterologous mixtures such as plasma and tissue extracts, needs to take into consideration the problems of cross-reactivity which will occur particularly with highly multiplexed assays. Antibodies can be surprisingly cross-reactive, which in the high throughput microarray field can render results misleading or, at worst, useless. Successful multianalyte analysis will therefore require careful screening of each polyclonal antiserum, hybridoma or recombinant clone for cross-reactions against all antigens on the array. The use of combinations of antibodies against individual targets in sandwich assays should help to minimize cross-reactions, or the linkage to mass spectrometry to confirm the identity of bound ligands. As diagnostic devices, microarrays exploit the power of multiplexing simultaneous analyses of different samples and repeated analyses of the same samples in automated way. Diagnostics formats include arrays of antibodies, as in detection of cytokines, and antigens to detect serum antibodies in screens for infections, autoimmune diseases and allergies. Highly parallel analysis on arrays will allow determination of tumour markers in extracts with only a minimum of biopsy material, creating new possibilities for monitoring cancer treatment and therapy. Discovery of new autoantibody specificities is possible by screening patient sera against arrays of human proteins. The quantitative detection of proteins in cells and tissues and comparison in different conditions (health, disease, differentiation, drug treatment, etc) is a central aim of proteomics. The array format is well established for the rapid, global analysis of nucleic acids, as in the use of oligonucleotide and cDNA arrays for gene expression (transcription) profiling. Two-dimensional gel electrophoresis technology, on which most proteome profiling is based currently, is also limited in various ways, particularly, as stated above, in the difficulty of finding and quantitatively estimating low abundance proteins. For information about the expression of the proteome, protein and peptide arrays are becoming major tools, and the information that will be obtained from them in the future will complement transcriptional data. Capture arrays sensitively and accurately detect low levels of proteins with minimal technical know-how on the part of the user, and we can expect them to be used widely to measure differential protein expression. They will provide a powerful and reliable platform for extending molecular analysis beyond the limitations of DNA chips. On the other hand, it is difficult to store large-scale protein arrays in a functional state for long periods of time, due to the sensitivity and heterogeneity of proteins, unlike DNA that is a highly stable molecule capable of long-term storage. Therefore, an interesting concept nowadays is to make protein arrays directly from DNA, either co-distributed or pre-arrayed, using cell free protein expression systems, to create the proteins on the arrays on demand as and when required. Thus, in situ methods address the three main issues in protein array technology: efficient global protein expression and availability; functional protein immobilization and purification, and on-chip stability over time. The stability and lifetime of protein arrays in different formats needs to be considered. Detection methods are another important consideration, with requirements of sensitivity, accuracy and quantization over a wide range. The design of the array will be influenced by the readout system. Finally, standardization is an issue common to all high throughput technologies: the existence and development of many alternative formats and conditions inevitably leads to problems in comparison of results. Standards for protein arrays and a framework for their implementation will need to be established at an international level.