Study On Interaction Of Protein Subunits And Protein-micromolecular By Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a “soft” ionizationtechnology. During the ionization process, biomacromolecules and noncovalentcomplexes will maintain the natural structure and enable them to carry multiplycharge, so it could detect in the low-mass range. Meanwhile there are manyadvantages of ESI-MS, known as the “3s”: high speed, high sensitivity and highspecificity. Therefore, ESI-MS has been widely used in many biochemicalresearches and plays an important role in the study of noncovalent complexes.The instrument parameters of ESI-MS that had chosen for experiment willaffect the result seriously. Appropriate instrument conditions not only could ensurethe signal response but also maintain the natural conformation of protein and theircomplexes. Firstly, in this paper we study the influence of various instrumentparameters on the signal response of alcohol dehydrogenase (ADH). The resultsshow that we mainly observed monomer and dimer of ADH via ESI-MS, and thepolymer formed by ADH is affect by the content of organic solvent that in themobile phase. When there are thirty percent acetonitrile in the mobile phase, we canobserve a large proportion dimer and little tetramer. And then Nicotinamide adeninedinucleotide (coenzyme) and4-methyl pyrimidine (dehydrogenase inhibitor) wereselected as substrates to ADH, but there are not noncovalent complexes detected byESI-MS. These results indicate that the interaction between ADH and substrates areaffect by the formation of tetramer seriously.Secondly we constructed ultrafiltration mass spectrometry methodology coupleultrafiltration device with mass spectrometer to rapidly screen the ligands that couldbind on biomacromolecules. In comparison to other methods, ultrafiltration massspectrometry is more rapid, sensitivity and high-throughput. And open a newmethod for the screening of drugs for specific targeting proteins pilot, developmentof new drugs and design.Human serum albumin (HSA) and the extract of soy isoflavone powder areincubation at a fixed molecular ratio. The free ligands are eluted by organic solvent,meanwhile the specific binding of ultrafiltration membrane and operate error areavoided by control experiment. Lastly, the free ligands are quantify by ESI-MS.These results indicate that this methodology could rapidly screen dadzin, genistin,daidzein and genistein that extract from soy isoflavone powder which could binding on HSA. This methodology couple ultrafiltration device with LC-MS which couldavoid the interference of sample matrix, improve the selectivity and easy to operate.Meanwhile the advantage that enables to screen various ligands that could bind onbiomacromolecules at the same time make it is very suitable to screen various activecompounds that are extracted from natural products.