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Figure 9.14. Decode user interface demonstrating the Code-to-Structure step.
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peak, which is higher than the Percentage, this is considered a failure for the synthesis step. If one, two, or three tags are found, then the tags are placed in alphabetical order and then the tag code is searched in the Oracle R-Group table. If there is a match, then the AF number (AF is the Affymax compound numbering system) is retrieved for the R-group along with the member number. These tags are marked as used. The pool number is used to retrieve a building for spatially or partially encoded steps. The screen is updated to show the Decode information, as in Figure 9.14. The well colors in the plate graphical user interface are adjusted to re ect success or failure of the Decode. Obviously the control wells will always come out red, as they do not represent coded molecules. At this point in the analysis, the user can review the data for completeness and study the failures. Failures are usually due to multiple beads or no beads in a well. The Export Decode Peak Data feature exports to Microsoft Excel a worksheet that can be used to track quantitative results of the decode process. The worksheet includes building block identi ers and the amounts
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characterization of split-pool encoded combinatorial libraries
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of tag detected in each well. These results are used in quality control of bead differentiation and encoding. 9.3.3. Code to Structure
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Prior to structure generation, it is assumed that a generic structure describing the combinatorial library exists in the Affymax ACL database. The ACL database is an ISIS/Host v3. RCG database that contains generic structures described with root structures and corresponding named building blocks (see Figure 9.9). Here each building block has a previously assigned name (e.g., AF11534). After the decode is complete, the Code to Structure button appears as shown in Figure 9.14. This button causes the enumeration string for positive decodes (in this case ACL2784-AF11534-AF11534AF11534) to be generated for each positive well and appends them to the AFFYBANK_ACL_DECODE table. An Oracle procedure is then launched, and it generates the structure from each enumeration string. Structures for the positive decodes are generated and placed in an ISIS database. 9.3.4. Capture
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We use the Windows NT application, CAPTURE,32 to correlate the decoded structures with the mass spectrum from each well in library quality control. For the example peptide library we would pick ve beads from each of the six pools. Each bead yields a ligand LC/UV/MS data le and a decoding LC/UV/MS data le. The user interface in CAPTURE represents a 96 well microtiter plate, as shown in Figure 9.15. For the green wells the predicted structure from decoding has matched a spectrum from the LC/MS data. This application uses simple isotopic composition and electrospray ionization rule sets to predict mass spectra and judge the concordance of a structure-mass spectrum data set. The white well is the case where no structure is available because the decoding has failed. The red well is the case with a good decoded structure but the LC/MS data do not match. The data behind the green light of well A01 is shown as Figure 9.16. The ligand LC/UV/MS is conducted full scan, treating the ligand as an unknown. In the gure the top trace is a base peak chromatogram (BPC), the middle trace is the UV chromatogram, and the bottom trace is the spectrum of the major or expected compound. The weakness of these signals is typical for single-bead mass spectra. Although the nominal amount of ligand cleavable from a single bead is in the hundreds of picomoles, variations in synthetic yield, cleavage yield, and ionization ef ciency can cause several orders of magnitude differences in actual LC/UV/MS peak sizes.
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