The calculation of the turnover frequencies (TOFs) enables to show that, among all the samples studied, Ph-PMO-PrSO3H exhibit the higher catalytic activity.
Fig. S2 Topograph calculation of protein turnover rate and peak alignment for isotopomer abundance.
Calculation of the turnover rate (kcat) of the enzyme was not done due to the high level of recombinant glycosylation of CmINV, making accurate molecular weight determination misleading.
This is key to accurate calculation of protein turnover rates; other heavy isotope labeling approaches require complex calculations to indirectly estimate and compensate for changes in precursor pool enrichment (Price et al., 2010, 2012; Kim et al., 2012).
The Topograph software program was developed for the deconvolution and measurement of peptide isotopologue abundances from LC-MS chromatograms, and the calculation of peptide turnover rates, as previously described (18) (http://proteome.gs.washington.edu/software/topograph/).edu/software/topograph/
The topograph software program (http://proteome.gs.washington.edu/software/topograph/)(http://proteome.gs.washington.edu/software/topograph/) was developed for the measurement of peptide isotopologue abundances from LC-MS/MS chromatograms and the calculation of peptide turnover rates (Hsieh et al., 2012).
Measurement of isotopic (e.g., 2H, 13C, 15N, and 18O) enrichment and concentrations of over 140 intermediary metabolites by GC-MS, LC/MS/MS, and NMR for the calculation of turnover of carbohydrates, lipids, and proteins.
In numerous preparations of recombinant yeast ADH1 in our laboratory, we used titration with NAD+ in the presence of 10 or 100 mM pyrazole to determine the concentration of active sites for calculation of turnover numbers.
Using peptides attached with a fluorescent label allows detection and quantification of both forms and hence the calculation of the reaction turnover.
