Purification of Peptides in High-Complexity Arrays

Compared to the human genome, which is formed by approximately 25,000 genes, the human proteome comprises over a million functional proteins and thus represents a much higher diversity. Although genetic research provides valuable information on the proteins which can be translated, a great task in the future will be the exploration of the entire protein interaction network. Understanding protein interactions will greatly help scientists to identify the mechanisms behind fatal diseases such as cancer, AIDS, and tuberculosis and, hopefully, provide new cures. Hence, micro arrays containing proteins or small protein fragments in the form of peptides have become of great interest in proteomic research. Using these microarrays a large number of potential target molecules can be screened for interaction with a probe in a short time frame. However, protein and peptide micro arrays are still lagging behind oligonucleotide arrays in terms of density, quality and manufacturing costs. A new approach developed at the German Cancer Research Center (DKFZ) has improved the synthesis of high density peptide arrays. The current technology is capable of producing arrays with up to 40,000 different peptides per cm² by means of a micro particle-based solid phase peptide synthesis (mpSPPS). Similar to Ronald FRANK’s SPOT synthesis, the peptides are combinatorially synthesized directly on a solid support, whereby the exact location of each peptide is known. However, the in situ synthesis bears a conceptual disadvantage: The quality of the peptides is dependent on the efficiency of the synthesis. Inefficient coupling produces peptide fragments which are present in the resulting array among the desired full length peptides. Thus, this PhD thesis dealt with the improvement of the peptide quality of in situ synthesized micro arrays. The central achievement is a new method allowing for the fast one-step purification of entire peptide arrays without loss of resolution or spatial information. The key principle is the transfer of an in situ synthesized array to a gold-coated polyvinylidenefluoride (PVDF) membrane, onto which only full-length peptides are allowed to rebind via an N-terminal cysteine. Peptides are synthesized on a solid support by means of mpSPPS using the acid-labile RINK amide (RAM) linker as an anchor group which allows for cleavage and removal of side chain protecting groups in one-step. After the synthesis, the array is brought into direct contact with the gold coated PVDF membrane. The membrane is soaked in trifluoroaceticacid (TFA) transfer medium which immediately initiates the peptide release and at the same time catalyzes a thiol-gold bond formation. Specific transfer could be verified down to a resolution of 10,000 spots per cm². Only cysteine terminated peptides which represent the full-length array members were transferred, whereas other peptides and synthesis fragments were excluded. The fluorescence signals on the target membrane appeared to be strong and almost background-free. Furthermore, no lateral diffusion was observed, which provides access to high complexity and high-quality peptide arrays in an easy manner

Peptide arrays with a chip

Today, lithographic methods enable combinatorial synthesis of >50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. A combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A computer chip consecutively addresses the different charged particles to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids in one single coupling reaction to the support. The method should allow for the translation of entire genomes into a set of overlapping peptides to be used in proteome research