Matrix of 2′,4′,6′-trihydroxyacetophenone (THAP) in acetonitrile (20 mg/ml) was applied directly to the 1536-spot surface. The matrix was allowed to dry, and each spot was analyzed by MALDI-TOF MS using an AB Sciex 5800 series instrument. Captured metabolites of the cell-free reactions were identified by their mass shifts relative to the unreacted peptide. Analysis of more than 10,000 spectra collected was performed using in-house, Python-based software package to automate the integration of peaks of interest from the mass spectra dataset. The software calculates the area under the curve at any mass/charge ratio value given to it over a user-defined half-width mass window. For this work, a half-width mass window of 0.2 mass units was used across all spectra analyzed. For every spectrum in this dataset, the following peaks were integrated: 1371 Da (capture peptide), 1385 Da (normalization peptide), 1413 Da (Ac-CoA reaction product), 1515 Da (HMG-CoA reaction product), and 1443 Da (pyruvaldehyde adduct). In addition, for each peak analyzed, regions immediately flanking the peak were integrated to determine a local spectral baseline, which was subtracted from the peak area. For isotopic labeling experiments, the area under the curve for various isotope peaks was also collected.

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