Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 66
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‘DNA chip’ technology has successfully been applied to a wide varietyof biological systems and has resulted in significant insights into human diseasessuch as cancer [22, 113–118].Arrays for proteome analysis may be separated into two groups, protein arraysand antibody arrays. Protein arrays comprise a capture molecule other than anantibody whose physiological interaction with other molecules is the basis of thescientific interest. Thus protein arrays might be designed to study enzyme-ligandbinding, to investigate the activity of proteins of interest in the presence of otherbiological compounds or drugs, to determine functionally important protein-protein interactions, or to form the basis of a serodiagnostic chip [119–124].
They14.4 Protein Arraysmay also be used for high-throughput screening of antibody specificity, involvingthe arraying of potentially genome-wide sets of recombinant proteins [120, 125,126]. Antibody arrays, on the other hand, comprise a series of immobilised antibodies used primarily for relative protein expression analysis and thus may beseen as an alternative to 2DE [61, 120], although they may also be used to evaluate antibody specificity in a high-throughput manner [127].The basic requirements of antibody and protein arrays are essentially the same:i) specific recognition molecules must be generated and isolated; ii) the recognition molecules must retain their biological activity when immobilised in an addressable format; and iii) bound target must be detected. As described above,these requirements are readily satisfied for DNA arrays but protein capture molecules are considerably more difficult to generate in quantity and at a high level ofpurity, plus immobilisation may affect their ability to capture the target antigenwith high specificity and affinity.
Stoichiometric modification of the protein sample with fluorescent or other tags is difficult to achieve and may influence the interaction of the labelled protein with the capture molecule. Optimal conditions forbinding of target protein to the array may differ widely for each capture moleculebecause of their widely divergent physicochemical properties, introducing afurther level of complexity to the interpretation of binding patterns. Thus it is difficult to envisage protein or antibody arrays that are analogous to DNA arrayswith current technology, but developments that are occurring across many disciplines may eventually lead to more limited but nonetheless useful protein andantibody arrays.14.4.1Protein Recognition MoleculesThe range of molecules available to be exploited as protein recognition molecules(PRMs) is expanding as new technologies are being developed to replace or complement older, more established strategies, although few methods for generatingcapture reagents specifically for incorporation into arrays for protein expressionprofiling have been described [128, 129].
Whatever their nature, the PRMs to beincorporated into protein or antibody arrays need to meet various criteria: firstly,one has to have access to a process that can support the generation and selectionof antibodies to individual proteins and possibly each of their post-translationallymodified forms; the antibodies must be highly specific, that is they must be capable of recognising and distinguishing the individual protein when presented in acomplex mixture; the binding of the individual proteins within the mixture totheir cognate proteins must occur under similar conditions, that is with similar affinity, association constant etc; and the array must be amenable to prolonged storage without reduction in the reactivity of some or all of the capture reagents.The biological function of polyclonal and monoclonal antibodies is molecular recognition, so that they are perhaps the most obvious choice as PRMs.
They arecapable of exquisite distinction between closely related proteins, such as those differing by phosphorylation at a single site, and there are well-established proce-23924014 Protein Chip Technologydures for the generation of polyclonal and monoclonal antibodies in vivo [130,131].
However, the generation and screening of antibodies is very demanding interms of the requirement for pure protein and cross-reactivity with moleculesother than the target protein cannot be ruled out.The production of synthetic phage-displayed antibodies provides considerablescope for the generation of protein recognition molecules to support the development of ‘protein chips’. Recombinant phage technology was established in theearly 1980’s and has developed into a rapid and simple method to link phenotypeto genotype by inducing recombinant phage to express a protein of interest, suchas an antibody fragment, on the surface coat where it is available for functionaland binding assays. The advantages of antibody phage display over conventionalmonoclonal antibodies include the ability to humanise the antibodies for use inimmunotherapy and the generation of antibodies against self antigens as a sourceof potential anti-cancer reagents [132–139].
Problems associated with the technology include the low affinity expressed by many of the antibodies selected fromphage displayed libraries, although there is the opportunity to ‘mature’ the antibodies in order to increase their affinity by processes such as in vitro mutagenesisand error-prone PCR [140–142]. Other problems include the genetic instability ofsome of the libraries [142], and the fact that the selection procedure requires multiple rounds of panning against the cognate protein.
As with monoclonal antibodies, selection of phage displayed antibodies requires significant quantities of puretarget protein, and unique specificity for the target cannot be assured.Various methods have been developed that employ peptides as either the capture reagent, or as a readily available source of target antigen for screening PRMs.Thus, random peptide aptamers may be introduced into cells in order to inhibitintracellular functions [143, 144], or peptides may be synthesized in situ to generate arrays for screening for interaction partners [145, 146], and the latter are likelyto be more flexible in terms of the range of specificities that may be achievedthan an array based on, for instance, monoclonal antibodies. However, their usefulness may be restricted when functional interactions are based on conformational rather than linear epitopes.
Polysome display involves the in vitro expression of a peptide and the stabilisation of the interaction between the ribosome,the mRNA and the peptide being expressed, providing a link between the peptideand its encoding gene. This allows multiple rounds of screening to be performedwithout the prior availability of large quantities of pure protein [147, 148].
The potential also exists to immobilise polysomes in an array format via molecular modification of the complexes with a DNA linker.Proteins are not the only PRMs that might be used for ‘protein chips’. Anygroup of molecules that can specifically recognise individual proteins with appropriate affinity and avidity and can do so in an immobilised form would be suitable.
Indeed, there are disadvantages to the use of proteins as recognition molecules, the most obvious ones being that the chips might suffer from poor storageproperties and that the presence of any protease activity within the protein mixtures to be analysed may ‘degrade’ the chip. Nucleic acid aptamers are singlestranded oligonucleotides possessing high affinity for conformational biomole-14.4 Protein Arrayscules, such as proteins.
Libraries of aptamers up to a few hundred nucleotides inlength may be immobilised to an appropriate surface for screening and may provide information on conformation, position of hydrogen bonds and other datawhich help to build up a three-dimensional model of the target protein [149–153].Nucleic acid aptamers also have advantages for ‘protein chips’ because the targetprotein does not need to be modified prior to capture, the aptamer itself acting asthe capture and detection reagent [149].Molecular imprinting involves the synthesis of artificial recognition sites on asurface by mimicking the shape of the template molecule in a polymeric film,thereby forming a molecularly imprinted polymer (MIP). It is proposed that anybiological situation in which shape plays a part, such as antibody-antigen interactions, substrate-enzyme binding and receptor-ligand binding, may be mimickedby MIPs [154–158].
Most of the studies performed to date with MIPs involve theimprinting of small molecules such as drugs and pollutants [155, 159, 160], butthere is great potential to create artificial capture surfaces that mimic antibodiesand receptors [157]. As with most of the PRMs described, a clear disadvantage ofMIPs is that their manufacture requires access to large quantities of the moleculeto be imprinted, plus the capture surfaces are in effect polyclonal and thereforethere may be problems with specificity.14.4.2Immobilisation of PRMsThe specific method employed to array and attach the PRMs to the array surfacewill obviously depend upon their nature.
The basic requirements of the arrayingprocedure are spatial definition, reproducibility, stability and retention of highspecificity and affinity. There are many different methods for arraying proteins(including antibodies) [161] and developments are continuously being made,many of them driven by the advances being made in biosensor technology [61,162–166].14.4.2.1 Arraying of Proteinaceous PRMsProteinaceous PRMs, be they monoclonal antibodies, phage displayed antibodiesor other polypeptides, will need to be attached to a suitable surface in a spatiallydefined manner.
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