U. Beutling, A. Dikmans, D. Oster, S. Thiele, A. Wiemann; with E. Wanker (MDC, Berlin)
Membrane-bound compound arrays manufactured by SPOT synthesis (see above) are ideally suited for a single user and can only be reused a few times. Furthermore, SPOT synthesis on porous membranes has its limitations when reducing the spot size below 1 mm and becomes costly and tedious when large numbers of copies of an identical array are required. We have therefore developed a special novel process for manufacturing and application of synthetic peptide/compound libraries in the form of chemical mini- or microarrays which keep the advantageous features of cellulose-bound probe molecules but allows massive miniaturization and multiplication (58).
The assembly of the peptide/compound macroarray follow essentially standard SPOT synthesis protocols with an array format adapted from the 384-well microtiter plates, except that a special, acid sensitive amino-cellulose membrane is used. Individual spots are separated post-assembly with the help of a 384-compatible punching device which delivers the cellulose-compound conjugate disc segments of 3 mm diameter into the wells of four 96-deepwell plates. Then, the discs are treated with a TFA cocktail containing >80% TFA plus scavengers, as used in routine solid phase peptide synthesis. This treatment solubilizes the support itself with the compounds still covalently attached and simultaneously cleaves acid sensitive side chain protecting groups. Precipitation with ether removes the bulk of acid together with the cleavage chemistry and the dried precipitate is then dissolved in DMSO. After appropriate dilution with DMSO, these solutions of compound-support conjugates are printed and adsorbed onto the target planar surfaces, usually glass microscope slides. We therefore call this process “spotting compound-support conjugates”: SC2. One standard cellulose spot yields 0.5 ml of DMSO stock solution from which nl to pl aliquots can be used to print up to 106 to 108 mini- to microarray copies.
A microarray of diketopiperazines prepared by new SC2-process on a glass microscope slide is probed with Calmodulin (CaM) in the absence (EGTA, left) and presence of calcium ions yielding two new ligands for CaM. W7 is a known small molecule ligand for CaM; biotin serves as control and is detected by addition of labelled streptavidin to the sample.
Experiments like profiling serum collections for humoral immune responses with peptide maps of antigens or profiling recombinant protein extracts with generic compound libraries are typical examples that require hundreds of array copies. For example, we have synthesized and printed about 3 000 overlapping 15mer peptide fragments covering the major virulence proteins of Yersinia enterocolitica for the immune-profiling of pathogen challenged mice over time course of immune response and genetic backgrounds for the EUMORPHIA project. Our small molecule microarrays are applied to systematically screen for small molecule ligands of hundreds of human proteins involved in disease related cellular processes (www.smp-protein.de). The corresponding patent could be licensed to Intavis AG (Cologne) and first products are already on the market.