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Data Reduction and Automated Interpretation of GPC Spin Column/ESI-MS Data

The interpretation of the large amounts of data generated in a screening campaign cannot be performed manually. Customized software has been designed to automatically evaluate the data and search for the compounds that non-covalently bind to the protein [21, 22]. For flow injection ESI-MS data of GPC spin column eluates of ten component mixtures, the following steps were taken to automatically interpret the data. The raw data were combined, smoothed, background

Fig. 2.9 Automation software for data reduction and analysis of GPC spin column/ESI-MS drug screening data. (A) Screenshot of the data analyzer program for combining, smoothing, background subtracting and centering of raw mass spectral data. (B) Algorithm for background subtraction of the previous spectrum from the foreground spectrum including the normalization of the chemical noise between the two spectra.

Fig. 2.9 Automation software for data reduction and analysis of GPC spin column/ESI-MS drug screening data. (A) Screenshot of the data analyzer program for combining, smoothing, background subtracting and centering of raw mass spectral data. (B) Algorithm for background subtraction of the previous spectrum from the foreground spectrum including the normalization of the chemical noise between the two spectra.

subtracted and centroided as illustrated in the screenshot of the data analyzer program (Fig. 2.9A). To remove the chemical background from the spectrum of interest (foreground spectrum), the prior sample analyzed in the screening campaign was used as the background spectrum. The background spectrum was subtracted from the foreground spectrum after applying a multiplication factor to the background spectrum to normalize the chemical noise, as described in the background subtraction algorithm (Fig. 2.9B). Figure 2.10 illustrates the application of the chemical noise background subtraction algorithm to an eluate of a ten-component mixture. For the components in the mixture, the computed [M+H]1+ values are matched with the observed values, the S/N ratios calculated for the observed ions and the theoretical isotopic distribution calculated and matched to the observed distribution. Each of these calculations are scored and weighted for

Fig. 2.10 Application of the background subtraction algorithm between two consecutively acquired ESI mass spectra from GPC spin column eluates of ten-component mixtures incubated with RGS4 protein. (A) Foreground ESI mass spectrum for the GPC spin column eluate of a ten-component mixture incubated with RGS4 protein from well A1. (B) Background ESI mass spectrum corresponding to the GPC spin column

Fig. 2.10 Application of the background subtraction algorithm between two consecutively acquired ESI mass spectra from GPC spin column eluates of ten-component mixtures incubated with RGS4 protein. (A) Foreground ESI mass spectrum for the GPC spin column eluate of a ten-component mixture incubated with RGS4 protein from well A1. (B) Background ESI mass spectrum corresponding to the GPC spin column eluate of a ten-component mixture incubated with RGS4 from the well analyzed prior to well A1. (C) The foreground ESI mass spectrum for well A1 after subtraction of the background spectrum using the background subtraction algorithm described in Fig. 2.9B. Note the appearance in the subtracted mass spectrum of peaks corresponding to small molecules non-covalently bound to the RGS4 protein, indicated with bold arrows.

each compound in the mixture, and if the total score is greater than a predicted threshold value, the component is considered a ''hit''. Generally, a scored S/N ratio greater than 25 is considered a ''hit''. An example of an automatically generated drug screening report is illustrated in Fig. 2.11.

Fig. 2.11 An automatically generated ''drug screening ESI-MS report'' for the ESI background subtracted mass spectrum, illustrated in Fig. 2.10C, obtained from the GPC spin column eluate of the ten-component mixture incubated with RGS4 protein from well A1. Note that the highlighted components are the three compound hits that non-covalently bind to the RGS4 protein.

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