What ist SPOT synthesis? The SPOT method was developed by Ronald Frank for simultaneous multiple peptide synthesis on separate sites on a homogeneous membrane carrier. The principle of the technique is to dispense small droplets of preactivated amino acid derivatives onto a predefined array of positions on a porous membrane. The droplets get absorbed and form individual reaction compartments for chemical reaction in solid phase synthesis. A great number of distinct spots can be arranged on a large membrane sheet and each of these is individually addressable by manual or automated delivery of the respective reagent solution. The number of spots per area and the spot size are determined by the absorptive capacity of the membrane and the volume of the drops. According to the specific functionality of the matrix, the spot size correlates with the particular scale of the synthesis. The membrane originally used by Ronald Frank is still the most popular support, made from derivatized cellulose filter paper.
Which steps are automated? The SPOT method was originally developed as a manual method and steps common to all spot reactors are carried out manually by washing the entire membrane with respective reagents and solvents. The most tedious step is spotting of individual activated amino acids to the reactive areas in patterns which change in each cycle. Automation of this step made the method applicable to a much wider range of experiments as it cuts down the time of work involved and allows to create much larger arrays with smaller volumes than can be handled manually. It is now possible to synthesize several thousand peptides in a single run. Automated deposition of amino acids also allows for easy double or triple couplings which improves the synthesis quality. Bulk treatments of the membrane like washing and deprotection are difficult to automate and are therefore still carried out manually.
What is the basic procedure? Peptide synthesis is a cyclic procedure with a number of steps carried out for each amino acid addition. Out of these steps only the delivery of activated amino acids to the membrane is automated by the AutoSpot. This step is actually the most tedious and error-prone one of the entire procedure.
Step 1: Fmoc deprotection Step 2: DMF wash Step 3: Ethanol wash Step 4: Air drying of the membrane Step 5: Spotting of activated amino acids and waiting for reaction time Step 6: Capping, optional Step 7: DMF wash Repeat synthesis steps 1-7 until the desired peptides have been assembled.
After peptide assembly the side chain protection groups must be removed in a different protocol using a mixture of trifluoroacetic acid (TFA) in dichloromethane (DCM) and appropriate scavengers.
What can be done with the AutoSpot? The AutoSpot was developed to reliably deliver amino acid derivatives to synthesis arrays on membranes. A pipetting robot under the control of a PC-based software now delivers volumes down to 100 nl to up to a few thousand individual spots. Four membranes of microtiter plate format can be mounted on the work area. Standard grids are 96 or 384 positions, but densities up to about 1000 spots per membrane have been achieved. The regular grid can be freely defined. Up to 44 amino acid derivatives can be used which allows for use of natural and non-natural amino acids in one run.
How much amino acid is needed for SPOT synthesis? One set of amino acid cartridges usually lasts for a synthesis of 4 membranes with 10-12mer peptides. The exact consumption depends on the sequences, spot size and protocol (single/double/triple coupling). Make sure to have at least another set available before starting the synthesis.
How much peptide is in a spot? The membranes we supply are derivatized to a loading of about 300-400 nmol/sqcm free amino groups. The loading per spot can be calculated from the area using this value. A spot of 6 mm diameter would have about 80 nmol, a spot of 3 mm diameter about 20 nmol of peptide.
Which chemistry is used? The synthesis is carried out using Fmoc-protection chemistry on membranes made of pure, derivatized cellulose. The amino acid building blocks are derivatives which are protected at their amino terminus by 9-fluorenyl-methoxycarbonyl (Fmoc). The protection group ensures, that in each step only a single building block is coupled to each growing peptide chain. Trifunctional amino acids also carry a side chain protection group. The Fmoc group must be removed in each synthesis cycle whereas the side chain protection groups are taken off at the end of the synthesis. In situ activation of the amino acid derivatives is performed by DIC/HOBt, which leads to rapid activation and coupling. A membrane can be used to synthesize a large number of individual peptides which can be screened for biological activity by a Western-blot-like assay. Alternatively, the spots are cut out and cleaved separately from the support if a suitable linker had been introduced prior to the synthesis.
Is it possible to make soluble peptides with the AutoSpot? Several people have worked on cleaving peptides from the SPOT membrane with different success. The major problem was always to get rid of the side chain protection groups while having the peptides still attached to the cellulose. Ronald Frank at GBF in Braunschweig, Germany, developed safety-catch linkers for this and he should have the best expertise. There is also a methods paper by Holger Wenschuh at Jerini Bio Tools, Berlin, which contains a number of protocols.
Is the protocol fool-proof for non-chemists? The protocol in the manual provides a detailed step-by step instruction for a standard scale synthesis. Some calculation is still required by the user, but once a specific protocol has been established at a facility this is hardly ever changed. We recommend a textbook on peptide chemistry for background reading. There should be someone around with a thorough understanding what the chemistry is about and what problems could arise. QC procedures are just mentioned as references without details.
Is it possible to synthesize PNA arrays with the AutoSpot? PNA is a very interesting DNA analog based on amino acid like building blocks. PNA spot arrays can be used for DNA hybridization. For protocols, please refer to papers published by Jörg Hoheisel et al.
Can the AutoSpot be used to make fluorescently labelled peptides? Some users have made fluorescently labelled peptides, and coupling of Rhodamine to some control sequences is also a nice way to monitor the synthesis. However, due to the open handling we would expect serious problems with photobleaching.
Can the peptide SPOT arrays be used for protease assays? Protease assays have been very difficult and only a very small fraction of the peptide on the paper is accessible for cleavage by enzymes. If is not possible to measure disappearance of labelled material but only the cleaved products themselves. Therefore the spots must be cut out before the assay which is quite cumbersome.
Can the peptide SPOT arrays be used for kinase assays? Kinase assays are an ideal application for peptide SPOT arrays as the labelled paper can easily be exposed to X-ray film for evaluation. There are several papers in the literature about this kind of assay.
