The rolC gene increases caffeoylquinic acid production in transformed artichoke cells.

Caffeoylquinic acids are found in artichokes, and they are currently considered important therapeutic or preventive agents for treating Alzheimer's disease and diabetes. We transformed artichoke [the cultivated cardoon or Cynara cardunculus var. altilis DC (Asteraceae)] with the rolC gene, which is a known inducer of secondary metabolism. High-performance liquid chromatography with UV and high-resolution mass spectrometry (HPLC-UV-HRMS) revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and chlorogenic acid. The rolC-transformed calli contained 1.5% caffeoylquinic acids by dry weight. The overall production of these metabolites was three times higher than that of the corresponding control calli. The enhancing effect of rolC remained stable over long-term cultivation.

Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured and denatured protein molecules are equilibrated at easily detectable ratios, namely 1:1. It also requires that closed and open states have lifetimes measurable in the time frame of the HDX experiment. Because both conditions are met by PEPS, these measurements can provide an accurate assessment of closed/open state populations and thus protein folding energies/rates.

Comparison of Two ESI MS Based H/D Exchange Methods for Extracting Protein Folding Energies.

In this report, the model proteins staphylococcal nuclease and ubiquitin were used to test the applicability of two new hydrogen/deuterium exchange (HX) electrospray ionization mass spectrometry (ESI-MS) methods for estimating protein folding energies. Both methods use the H/D exchange of globally protected amide protons (amide protons which are buried in the hydrophobic core) to elucidate protein folding energies. One method is a kinetic-based method and the other is equilibrium-based. The first method, the HX ESI-MS kinetic-based approach is conceptually identical to SUPREX (stability of unpurified proteins from rates of H/D exchange) method but is based on ESI-MS rather than MALDI-MS (matrix assisted laser desorption mass spectrometry). This method employs the time-dependence of H/D exchange using various denaturant concentrations to extract folding energies. Like SUPREX, this approach requires the assumption of EX2 exchange kinetics. The second method, which we call a protein equilibrium population snapshot (PEPS) by HX ESI-MS uses data collected only for a single time point (usually the shortest possible) to obtain a snapshot of the open and closed populations of the protein. The PEPS approach requires few assumptions in the derivation of the equations used for calculation of the folding energies. The extraction of folding energies from mass spectral data is simple and straightforward. The PEPS method is applicable for proteins that follow either EX1 or EX2 HX mechanisms. In our experiments the kinetic-based method produced less accurate ΔG(H(2)O) and m(GdHCl) values for wild-type staphylococcal nuclease and mutants undergoing H/D exchange by EX1, as would be expected. Better results were obtained for ubiquitin which undergoes HX by an EX2 mechanism. Using the PEPS method we obtained ΔG(H(2)O) and m(GdHCl) values that were in good agreement with literature values for both staphylococcal nuclease (EX1) and ubiquitin (EX2). We also show that the observation of straight lines in linear extrapolation method (LEM) plots is not a reliable indicator of the validity of the data obtained using the LEM approach.