Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
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  Affinity Proteomics  -  Protein Microarrays
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   Protein Microarrays

   Peptide Microarrays
Antibody
Microarrays

     In situ expression

     Personalised proteomics
     Peptide array in situ synthesis

    Affinomics
     T. brucei protein array
     Epitope mapping


    Single-molecule detection
     Interaction studies
     TargetBinders consortium
Archive
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As an immediate 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. Post-transcriptional control of protein translation, post-translational modifications as well as protein degradation by proteolysis, for example, have profound effects at the functional level. Estimations suggest that there are more than 200 types of protein modification. Its proportion and importance is reflected by the fact that 5% to 10% of mammalian genes encode for proteins that modify other proteins.
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The complexity in the human proteome is expected to range from a hundred thousand to several million different protein molecules. With respect to data interpretation, the situation is additionally complicated by the facts that not for every protein of multicellular organisms the function is known and that there may be different functions dependent on structure variations or interacting partners. In addition, 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. Nevertheless, many other, possibly even more powerful methods are prerequisite to approaching the world of protein analysis in a manner similar to what is already possible for studies at the level of nucleic acids, and beyond.
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Protein microarrays have an enormous potential of developing into a tool that will allow 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 a molecular understanding the biology of cells, tissues or whole organisms and has a broad biotechnical and medical potential. Utilising recently developed processes, we perfom such analyses on a large-scale with nevertheless high reproducibility, a near-single-molecule sensitivity, and an accuracy that is as good as or even superior to ELISA-based assays.
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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 a cell-free transcription/translation process on the microarrays, starting from full-length cDNAs. Protein interaction of all kinds as well as the influence of co-factors such as small molecules can be studied this way. The largest protein array produced so far contains some 14,000 individual proteins. The current set-up was and is used in various projects for actual measurements, frequently combining the information on protein levels with other data.
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Personalised proteomics:

In a recently finished 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 samll compounds - can be studied in a quantitative manner.
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Antibody microarrays:
Utilising antibody microarrays, we pursue the analysis of studying variations in actual protein abundance, isoform occurrence and other structural variations, in particular in cells, tissues and body liquids. Basic technical processes were studied in much detail, such as appropriate surfaces, the effect of kinetics and mass transport and labelling procedures as well as many other aspects in order to establish a working system. 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 company partners as well as EU-funded initiatives that aim at the creation of well-characterised and specific antibodies or other binders (e.g., Affinomics). In addition, improvements of the 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, either by actual counting of individual molecules or by an analysis of dissociation parameters.







Syafrizayanti et al. (2017) Sci. Rep. 7, 39756. pdf icon
Schmidt et al. (2011) J. Prot. Res. 10, 1316. pdf icon



Kamhieh-Milz et al. (2016) J. Proteomics 150, 74-85. pdf icon
Schröder et al. (2011) Protein Micoarrays - Meth. Mol. Biol., 203. pdf icon



Bakdash et al. (2016) Cancer Res. 76, 4332-4346. pdf icon
Alhamdani & Hoheisel (2011) Mol. Anal. & Genome Disc., Wiley, 219.




Loeffler et al. (2016) Nature Comm. 7, 11844. pdf icon
Sill et al. (2010) BMC Bioinformatics 11, 556. pdf icon



Sill et al. (2016) Microarrays 5, 19. pdf icon
Alhamdani et al. (2010) Proteomics 10, 3203. pdf icon



Kibat et al. (2016) New Biotechnol. 33, 574-581. pdf icon
Schröder et al. (2010) Antibody Engineer., Vol. 2, Springer, 429. pdf icon



Bal et al. (2016) Br. J. Haematology 4, 602-615. pdf icon
Alhamdani et al. (2010) J. Prot. Res. 9, 963. pdf icon



Nijaguna et al. (2015) J. Proteomics 128, 251-261. pdf icon
Schröder et al. (2010) Mol. Cell. Prot. 9, 1271. pdf icon



Mock et al. (2015) Oncotarget 6, 13579-13590. pdf icon
Gloriam et al. (2010) Mol. Cell. Prot. 9, 1. pdf icon



Betzen et al. (2015) Proteomics Clin. Appl. 9, 342. pdf icon
Alhamdani et al. (2009) Genome Med. 1, 68. pdf icon



Bradbury et al. (2015) Nature 518, 27. pdf icon
Börner et al. (2009) BioTechniques 46, 297. pdf icon



Hoheisel (2014) labor&more 10/14, 10. pdf icon
Taussig et al. (2007) Nature Meth. 4, 13. pdf icon



Srinivasan et al. (2014) Proteomics 14, 1333. pdf icon
Kusnezow et al. (2007) Proteomics 7, 1786. pdf icon



Syafrizayanti et al. (2014) Exp. Rev. Prot. 11, 107.
pdf icon
Kusnezow et al. (2006) Mol. Cell. Prot. 5, 1681. pdf icon



Marzoq et al. (2013) J. Biol. Chem. 288, 32517. pdf icon
Angenendt et al. (2006) Mol. Cell. Prot. 5, 1658. pdf icon



Lueong et al. (2013) J. Prot. Bioinf. 07, 004. pdf icon
Kusnezow et al. (2006) Proteomics 6, 794. pdf icon



Schröder et al. (2013) Proteomics Clin. Appl. 7, 802. pdf icon
Kersten et al. (2005) Expert Rev. Proteomics 2, 499. pdf icon



Hoheisel et al. (2013) Proteomics Clin. Appl. 7, 8. pdf icon
Kusnezow & Hoheisel .(2003) J. Mol. Recognit. 16, 165. pdf icon



Alhamdani et al. (2012) J. Proteomics 75, 3747. pdf icon
Kusnezow et al. (2003) Proteomics 3, 254. pdf icon



Friedrich et al. (2011) Proteomics 11, 3757. pdf icon
Kusnezow & Hoheisel (2002) BioTechniques 33, 14. pdf icon















Personalised proteome analysis by means of protein microarrays made from individual patient samples


DNA sequencing has advanced to a state that permits studying the genomes of individual patients as nearly a matter of routine. Towards analysing at a tissue’s protein content in a similar manner, we established a method for the production of microarrays that represent full-length proteins as they are encoded in individual specimens, exhibiting the particular variations, such as mutations or splice variations, present in these samples. From total RNA isolates, each transcript is copied to a specific location on the array by an on-chip polymerase elongation reaction, followed by in situ cell-free transcription and translation. These microarrays permit parallel analyses of variations in protein structure and interaction that are specific to particular samples
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Apart from procedural information, details are provided on the overall quality of protein microarrays (e.g., percentage of full-length molecules and the structural integrity of proteins) and their perfomance in various forms of analysis.


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First panel: Principle and performance of the process of producing personalised protein microarrays. (a) Schematic illustration of the overall process.
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Second panel: Protein microarray quality. (b) Microarray with 2016 proteins that were expressed in situ; visualisation was by incubation with red and green labelled antibodies that recognise common N- or C-terminal epitopes, respectively. (c) The typical ratio is shown of foreground to background signals for the N- (left) and C-terminus (right); blue lines indicate regions of identical intensity. (d) Detection of the C-termini of the 2016 expressed proteins with Cy3-conjugated anti-V5 antibody; the horizontal line represents a signal of three standard deviations above background. (e) Determination of the amount of in situ synthesised GFP by comparison to spotted material of known concentration; twenty measurements each were done; the red line represents the average amount of synthesised protein plus/minus one standard deviation. (f) Microarrays of T. brucei proteins were incubated with the labelled, synthetic RNA sequences shown. The white circles highlight positive signals. On the lower array, also a second protein exhibited interaction. In (g), the interacting protein from the lower microarray was analysed in more detail in comparison to a derivative with one point mutation (Mut) and another, unrelated protein. The upper level shows the protein amounts, detected by antibody binding to the N-terminus. The lower panel shows binding of the synthetic RNA. Subsequent studies demonstrated a more than 3,000-fold difference in affinity to the specific RNA between wildtype and mutated protein. (h) Binding to 74 human transcription factors of a labelled synthetic DNA sequence representing the TERT promoter (left). Mutation of one base pair in the binding sequence led to a very different binding pattern (right).
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Third panel: Detection of proteins generated in situ from individual samples. (i) Quality assessment of on-chip PCR by oligonucleotide hybridisation; two oligonucleotides were used, labelled red or green, respectively, each binding to one of the DNA-strands. Typical results are shown with an oligonucleotide specific to only one PCR-product present in quadruplicate (left) and a simultaneous hybridisation with oligonucleotides to all PCR-products (right). (j) Fusion of the images obtained after an incubation with fluorescently labelled antibodies against N- (red signal) and C-terminal tags (green signal) of the expressed seven tumour marker proteins. Spots 8 and 9 were negative controls without DNA-template. (k, l) Protein detection with labelled antibodies that target proteins CDK2 and TP53, respectively. (m) Results obtained on arrays produced from tissue samples of individual patients. Normal = healthy pancreas; CP = chronic pancreatitis; PDAC = pancreatic ductal adenocarcinoma; MiaPaca-2 = PDAC cell line. All proteins were identified with a tag-specific antibody (left). Binding patterns obtained with two different, isoform-specific antibodies. One isoform of the RUNX1 protein was present in all samples (right); the other one was found in diseased material only (middle). (n) Ninety-six DARPin binders were expressed in situ, each in three copies. Tag-specific antibodies identified all binders (left). The other two panels (middle, right) show binding patterns obtained upon incubation with protein AKT3. The white frames indicate the 16 binders that were expected to interact with AKT3. Different washing stringency produced distinct variations in the binding patterns.


Syafrizayanti et al. (2017) Sci. Rep. 7, 39756. pdf icon











Combinatorial peptide synthesis with laser-based transfer of monomers

              
 

Laser writing is used to structure surfaces in many different ways in materials and life sciences. However, combinatorial patterning applications are still limited. In a collaborative project coordinated by colleagues at KIT, a method was developed for cost-efficient combinatorial synthesis of very-high-density peptide arrays with natural and synthetic monomers. A laser automatically transfers nanometre-thin solid material spots from different donor slides to an acceptor. Each donor bears a thin polymer film, embedding one type of monomer. Coupling occurs in a separate heating step, where the matrix becomes viscous and building blocks diffuse and couple to the acceptor surface. Furthermore, two material layers of activation reagents and amino acids can be deposited consecutively. Subsequent heat-induced mixing facilitates an in situ activation and coupling of the monomers. This allows to incorporate building blocks with click chemistry compatibility or a large variety of commercially available non-activated, for example, post-translationally modified building blocks into the array’s peptides with >17,000 spots per square centimetre.

Loeffler et al. (2016) Nature Comm. 7, 11844. pdf icon











image of two protein arraysschematic presentation of the processProduction of high-density protein-microarrays by cell-free in situ expression

             
  logo EU FP7 


Due to the success of DNA-microarrays and the growing numbers of available protein expression clones, protein microarrays become more and more popular for the high-throughput screening of protein interactions. However, the widespread applicability of protein microarrays for this and other applications is hampered by the large effort associated with their production. Beside the requirement for a protein expression library, the actual protein expression and purification represents bottleneck.
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As part of the EU-funded MolTools project, we established a process that allows the generation of protein microarrays from process by which proteins are expressed from unbound DNA template molecules on the microarray surface (or on any solid support). It comprises the spotting of DNA templates onto the surface and the transfer of a cell-free transcription and translation mix on top of the same spot in a second spotting run. Using wildtype GFP as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein is produced to yield signals that are comparable to 300 µg/ml of spotted protein. Plasmids as well as unpurified PCR-products can be used as templates and as little as 35 fg of PCR-product (~22,500 molecules) are sufficient for the expression of full-length proteins.
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We adapted the system to the high-throughput expression of libraries by designing a single primer pair harbouring promoter, ribosomal binding site and terminator sequences for an on the chip expression of a multitude of such PCR-products. Utilising full-length cDNA libraries of overall 16,000 human clones, we are producing such microarrays for various types of analysis.
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Utilising the capability of detecting the interaction of individual molecules and therefore being able to count the actual number of interacting molecules, we are able to perform interaction studies in a really quantitative manner.




Schmidt et al. (2011) J. Prot. Res. 10, 1316-1322. pdf icon


Angenendt et al. (2006) Mol. Cell. Prot. 5, 1658-1666. pdf icon


Sobek et al. (2006) Comb. Chem. High-Throughput Screening 9, 365-380. pdf icon


Kersten et al. (2005) Expert Rev. Proteomics 2, 499-510. pdf icon


Angenendt, P. (2005) Drug Discovery Today 10, 503-511. pdf icon



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