LIPID ANALYSIS BY QUANTITATIVE THIN-LAYER CHROMATOGRAPHY.

Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.

Chemistry Techniques, Analytical↗

PubMed2005

Experimental configurational landscapes in aqueous solutions.

Structures and interactions between molecules in solution are modulated by the solvent. Changes in solvent conditions can lead to structural changes and transitions such as the assembly processes seen in micelle formation and protein folding. In the case of even quite complex liquid systems, we can now explore experimentally the configurational energy landscapes that underlie these processes. Using an aqueous solution of an amphiphile as an example, the structural transitions induced by changes in temperature, concentration and added salt are examined at the molecular level, and some critical regions of the landscape identified. Moreover, the potentials of mean force that quantitatively describe the solvent-modulated interaction between molecules in solution can now be experimentally accessed.

Chemistry Techniques, Analytical↗

PubMed2005

Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched-flow setup.

Amide hydrogen exchange (HX) in combination with mass spectrometry (MS) is a powerful tool to analyze the folding and dynamics of proteins. In the traditional methodology the exchange time is controlled by manual pipetting, thereby limiting the time resolution to several seconds. Some conformational changes in proteins, however, occur in the subsecond time scale, making it desirable to perform HX at shorter time intervals down to the limit set by the intrinsic chemical exchange rate. We now report the development of the first completely on-line quenched-flow setup that allows the performance of HX experiments in the 100-sec to 30-sec time scale, on-line proteolytic digestion using immobilized proteases, rapid desalting, and MS analysis. We show that conformational fluctuations in the range of seconds can be detected and protection factors as small as 10 reproducibly determined. Using this setup we investigated the conformational properties of Escherichia coli heat-shock transcription factor sigma32 free in solution. Our results indicate that the C-terminal sigma4 domain of sigma32, which is responsible for the recognition of the -35 region of heat shock promoters, contains more extensive secondary structure than expected when compared with the structure of the homologous sigma-factor sigmaA in complex with the RNA-polymerase. This setup should be very useful for a more accurate analysis of structural motions in proteins in the subsecond to second time scale relevant to allostery and enzyme function.

Chemistry Techniques, Analytical↗