Angelika Lueking

Protein arrays for gene expression and molecular interaction screening.

The array format has revolutionised biomedical experimentation and diagnostics, enabling ordered high-throughput analysis. During the past decade, classic solid phase substrates, such as microtitre plates, membrane filters and microscopic slides, were turned into high-density, chip-like structures. The concept of the arrayed library was central to this development which now extends from DNA to protein. The new and versatile protein array technology allows high-throughput screening for gene expression and molecular interactions. As a major platform for functional genomics, it is already on its way into medical diagnostics.

A Nonredundant Human Protein Chip for Antibody Screening and Serum Profiling

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm2 on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-μl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis6, anti-GAPDH, and anti-HSP90β antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.

Large-scale plant proteomics.

Large-scale and high throughput approaches increasingly play an essential role in the study of biological systems, which are per se highly complex. Therefore, they need to be examined by these extensive methods to receive information about the large genomic and proteomic networks. In plant biology, this purpose has a strong support through the accessability of the complete genome sequence of the model plant Arabidopsis thaliana. This brief review intends to focus on the basics and the state-of-the-art of these high-throughput technologies and their application to plant proteomics. It describes protein microarrays, the use of antibodies, 2-DE and MS methods and the yeast two hybrid system, which are emerging as the major technologies for plant proteomics.

Protein biochips: a new and versatile platform technology for molecular medicine

The human genome has been sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. High-throughput technologies such as DNA microarrays have been developed for the profiling of gene expression patterns in whole organisms or tissues. Protein arrays are emerging to follow DNA chips as possible screening tools. Here, we review the generation and application of microarray technology to obtain more information on the regulation of proteins, their biochemical functions and their potential interaction partners. Already, a large variety of assays based on antibody-antigen interactions exists. In addition, the medical relevance of protein arrays will be discussed.

High-throughput protein arrays: prospects for molecular diagnostics

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations.

Profiling of Alopecia Areata Autoantigens Based on Protein Microarray Technology

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Protein Array Technology: The Tool to Bridge Genomics and Proteomics

The generation of protein chips requires much more efforts than DNA microchips. While DNA is DNA and a variety of different DNA molecules behave stable in a hybridisation experiment, proteins are much more difficult to produce and to handle. Outside of a narrow range of environmental conditions, proteins will denature, lose their three-dimensional structure and a lot of their specificity and activity. The chapter describes the pitfalls and challenges in Protein Microarray technology to produce native and functional proteins and store them in a native and special environment for every single spot on an array, making applications like antibody profiling and serum screening possible not only on denatured arrays but also on native protein arrays.

Protein Array Technology Potential Use in Medical Diagnostics

The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome expression profiling is an important tool for functional genomic studies. Automated technology allows high-throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization or sequencing, hence creating a direct link between gene catalogues and functional catalogues. Microarrays can now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become amenable to database integration including all steps from DNA sequencing to functional assays of gene products.

Serum‐autoantibodies for discovery of prostate cancer specific biomarkers

The currently used prostate cancer serum marker has a low cancer specificity and improved diagnostics are needed. Here we evaluated whether autoantibodies are present in sera of prostate cancer patients and whether they are useful diagnostic markers for prostate cancer.

Off-target activity of TNF-α inhibitors characterized by protein biochips

Tumor necrosis factor-alpha inhibitors are widely and successfully used to treat rheumatic diseases. However, significant side effects have been reported. To detect the potential off-target activities of such inhibitors we characterized two therapeutic antibodies (adalimumab, infliximab) and one receptor fusion protein (etanercept) on protein biochips (UNIchip® AV-400) containing a printed serial dilution of tumor necrosis factor-alpha and about 384 different human proteins. Etanercept binds to ten proteins (affinity: 20–33% of tumor necrosis factor-alpha recognition), and six of these proteins are related to ribosomal proteins. Interestingly, adalimumab binds to the same six proteins related to ribosomal proteins (affinity: 12–18%) as well as to four proteins crucially involved in ribosomal protein synthesis. Alignment of protein sequences indicates no significant sequence homology between these ten proteins bound by the biological drugs with the highest off-target activities. Taken together, our in vitro results demonstrate that a significant number of proteins are recognized by tumor necrosis factor-alpha inhibitors and are related to ribosome biogenesis.