As a consequence of large-scale genomic sequencing, strong interest has emerged in analysing the function of the DNA-encoded information on a similarly global scale. However, many aspects of modulation and regulation of cellular activity cannot be investigated at the level of nucleic acids but require an analysis of the proteome.

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The complexity in the human proteome is expected to range from one hundred thousand to several million different protein molecules, all based on the about 22,000 protein-encoding genes. The situation is additionally complicated by the facts that not for every protein of multicellular organisms the function is known and that a protein may have different functions dependent on structure variations, interacting partners, location and time of expression. Also, the dynamic range of protein expression is very large indeed.
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Various mass-spectrometry-based processes exist for a powerful analysis of proteins of an organism or tissue. Also, assays such as yeast-two-hybrid analyses in all their facets permit global studies for the identification of interaction partners. As a third approach, affinity proteomics has an enormous potential in a global characterisation of molecule mixtures at the protein level. Knowledge of genomic sequences and transcriptional profiles do not suffice for a reliable description of actual protein expression, let alone an analysis of protein structures and biochemical activities or a quantitative examination of protein-protein interactions. This kind of information, however, is crucial for an understanding of the molecular biology of cells, tissues or whole organisms and has a broad biotechnical and medical potential. We perfom such analyses on a relatively large-scale with nevertheless high reproducibility, a near-single-molecule sensitivity, and an accuracy that is superior to ELISA-based assays.

Antibody microarrays:
Utilising antibody microarrays that currently consist of some 3,400 antibodies, we pursue the analysis of studying variations in actual protein abundance, isoform occurrence and other structural variations. Basic technical processes are understood in much detail, such as the choice of appropriate surfaces, the effect of kinetics and mass transport and labelling procedures as well as many other aspects. Detailed protocols are available that allow reproducible and reliable analysis of expression variations on complex protein extracts from tissues, cells or body liquids down to attomolar concentrations. Antibody generation and selection was and is performed in collaborations with companies as well as academic partners, partly within consortia that aim at the creation of well-characterised and specific antibodies or other binders (e.g., Affinomics). In addition, improved preparation of protein extracts proved crucial for success. The current set-up was and is used in various projects, frequently combining the information on protein levels with other data. Also, quantification of the results is performed.

Protein microarrays:
We utilise protein microarrays containing mostly full-length molecules for the investigation of protein-interactions in a quantitative manner. Microarray production is done by in situ synthesis by an in vitro transcription and translation process on the microarrays, starting from full-length cDNAs or gene-specific PCR-products. Protein interaction of all kinds as well as the influence of co-factors such as small molecules are studied this way. The most complex protein array produced so far contained some 14,000 individual proteins. The set-up is used in various projects and on the proteome of several organisms.

Personalised proteomics:
In a recently completed technical development, we added to the in situ protein production a process that allows to present on the microarray the proteins in exactly the conformation as they occur in tissues or other samples of individual patients, reflecting all mutations or splice variants that are specific for the particular sample. Thereby, particularly the effects of individual variations on protein interaction - with other proteins, nucleic acids or small compounds - can be studied in a quantitative manner.
 

As a multiplexing technique, antibody microarrays facilitate the highly parallel detection of thousands of different analytes from very small sample volumes of only few microliters. This is combined with a high sensitivity in the picomolar to femtomolar range, which is similar to the sensitivity of ELISA, the gold standard for protein quantification. In order to obtain such sensitivities in a robust and reproducible manner for complex analytes, it is essential to use an optimised experimental layout, sample handling, labelling and incubation as well as defined data processing steps.
 
Based on earlier work, we continuously developed the processing of microarrays and protein samples. In the publications listed below, our antibody microarray protocols for multiplexed expression profiling studies are described in detail; they permit the analysis of the abundance of very many proteins in plasma, urine, cell and tissue samples.