2640-07-5Relevant academic research and scientific papers
Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry
Cheng, Si,Wu, Qiuhua,Xiao, He,Chen, Hao
, p. 2338 - 2344 (2017)
Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis-Menten constants Km for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine Km and turnover number kcat for trypsin digestion of angiotensin II (Km and kcat are determined to be 6.4 mM and 1.3 s-1, respectively).
Modelling of prebiotic synthesis and selection of peptides under isothermal conditions and thermal cycling mode
Demina,Kononikhin,Laptev,Khodonov,Nikolaev,Varfolomeev
, p. 422 - 441 (2013/06/27)
The model peptide synthesis from mixtures of amino acids was carried out under the thermal cycling and isothermal modes. The compositions of the obtained mixtures of products and the primary amino acid sequence of the synthesized peptides were determined by Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry in combination with high-performance liquid chromatography with the application of de novo sequencing of the synthesized products. The processes of abiogenous synthesis of peptides were shown to occur under relatively mild temperature conditions and give a substantially less number of peptides as compared with the possible statistical set. The evolution of the system takes place in the process of the synthesis in solid phase with the disappearance of a series of the most unstable peptides. The selection process with the formation of complementary peptides takes place in peptide synthesis under the thermal cyclic mode.
