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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Technical Aspects
Peptide Microarrays
Biomedical Studies
Antibody Microarrays
   - Personalised proteomics
   - Peptide array in situ synthesis
   - Patient immunoprofiling     - Transcription factor binding
   - In situ expression protocol
   - Epitope mapping
   - Infection and cancer    - T.b. mRNA binding proteins

   - Single-molecule detection
   - TargetBinders consortium




Archive
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We utilise protein microarrays containing (mostly full-length) molecules for the investigation of protein-interactions. Microarray production is done by means of in situ synthesis in a cell-free transcription/translation process on the microarrays, starting from full-length cDNAs or PCR-products using gene-specific primers. Because of two subsequent spotting events, no contamination is possible between spots. Also, reagent consumption is kept to a minimum. Last, no particular attachment process is required; expressed proteins stick to their respective positions. Protein interaction of all kinds as well as the influence of co-factors, such as small molecules, on these events is studied this way. The complexity of the protein arrays produced so far ranges from a few hundred molecules to some 14,000 individual proteins. The set-up is used in various projects, frequently combining the obtained information with other data.
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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, the effects of individual variations on protein interaction - with other proteins, nucleic acids or small compounds - can be studied.
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Scheme of producing complex proteome microarray by in situ transcription & translation.
Testing for full-length products by using tag-specific antibodies (shown on the right) is done for few, randomly selected arrays only.

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Hufnagel et al. (2019) Bio-protocol 9, e3152. pdf icon






Hufnagel et al. (2018) Sci. Rep. 8, 7503. pdf icon
Lueong et al. (2013) J. Prot. Bioinf. 07, 004. pdf icon



Syafrizayanti et al. (2017) Sci. Rep. 7, 39756. pdf icon
Hoheisel et al. (2013) Proteomics Clin. Appl. 7, 8. pdf icon



Loeffler et al. (2016) Nature Comm. 7, 11844.
pdf icon
Schirwitz et al. (2013) Adv. Materials 25, 1598-1602. pdf icon



Kibat et al. (2016) New Biotechnol. 33, 574-581. pdf icon
Schirwitz et al. (2012) Biointerphases 7, 47. pdf icon



Lueong et al. (2016) Mol. Microbiol. 100, 457-471. pdf icon
Friedrich et al. (2011) Proteomics 11, 3757. pdf icon



Betzen et al. (2015) Proteomics Clin. Appl. 9, 342.
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Schmidt et al. (2011) J. Prot. Res. 10, 1316. pdf icon



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



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



Erben et al. (2014) PLOS Pathogens 10, e1004178. pdf icon
Angenendt et al. (2006) Mol. Cell. Prot. 5, 1658. pdf icon



Syafrizayanti et al. (2014) Exp. Rev. Prot. 11, 107. pdf icon
Kersten et al. (2005) Expert Rev. Proteomics 2, 499. pdf icon




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Immunoprofiling patients with Chlamydia infection and cervical cancer

In a collaboration with the DKFZ group of Tim Waterboer, we describe a method for identification of disease-related serum antibodies that are specific for infection and cancer, using Chlamydia trachomatis (Ct) as a complex model organism. Bacterial whole-proteome microarrays were generated using cell-free, on-chip protein expression. Expression constructs were generated by two successive PCR directly from bacterial genomic DNA. Bacterial proteins expressed on the microarray display antigenic epitopes, thereby providing an efficient method for immunoprofiling of patients. Antibodies from patient serum was analysed as shown in the scheme above. Through comparison of antibody reactivity patterns, we identified antigens recognized by known Ct-seropositive samples, and antigens reacting only with samples from cervical cancer patients. Large-scale validation experiments using high-throughput suspension bead array serology confirmed their significance as markers for either general Ct infection or cervical cancer, providing evidence for a role of Ct infection in the development of cervical cancer.

Hufnagel et al. (2018) Sci. Rep. 8, 7503. pdf icon
Hufnagel et al. (2019) Bio-protocol 9, e3152. pdf icon



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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.


Figure.
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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

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Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA binding proteome

Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function

Lueong et al. (2016) Mol. Microbiol. 100, 457-471.  pdf icon
Erben et al. (2014) PLOS Pathogens 10, e1004178pdf icon




Nine microarrays containing proteins expressed from 5 pg each of some 14,000 PCR-products.
The microarray-bound proteins were labelled with luminescent dye for quality control purposes










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









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