Charge-dependent Modulation Of Protein-metal Ion Interactions In Electrospray Ionization Mass Spectrometry

Protein-metal ion interactions have important physiological functions,including regulation of protein structure,storage of metal ions,transfer of electrons,transport of oxygen,and catalysis of reactions.At present,one-third of proteins need to interact with metal ions to perform biological functions.Studying these protein-metal ion interactions can help us to deeply understand senescence,apoptosis,and abnormal biological function of cells.Electrospray ionization mass spectrometry(ESI-MS)is currently used to analyze protein-metal interactions.Because of the gentle ionization process of ESI-MS,not only complete structure of protein macromolecule can be maintained,but also protein-metal ion interaction complex can be retained.Compared with UV,fluorescence,nuclear magnetic resonance(NMR)and other detection technologies,mass spectrometry can directly provide stoichiometry information of protein-metal ion interaction,and help to obtain the data such as binding constant and affinity in protein-metal ion interaction.In addition,ESI-MS can also be combined with liquid chromatography(LC),capillary electrophoresis(CE)and other technologies to obtain information on protein interactions in complex biological samples.However,there are some problems in ESI-MS detection,one of them is that ESI-MS can generate multiply charged protein ions.Although this feature allows protein peaks to appear in lower m/z range,allowing ESI-MS to be used for detection of la rger molecular weight proteins,different charge states may affect protein-metal ion interactions.For different charge states,three-dimensional structure of gas molecules,ionization efficiency,and mass spectrometry response factors of protein molecules are more or less different,which may lead to different combination ratio for different charge states in mass spectrum.This article systematically studies the effect of charge state on protein-metal ion specific interactions.It is believed that the difference in gas phase structure of proteins with different charge states leads to the change in their affinity for metal ions.We used techniques of photodissociation(PD)and hydroquinone exchange(HDX)to study gas phase structure of protein-metal ion complexes.Understanding the effect of charge state on protein-metal ion specific interactions can help us to obtain more real and reliable data when using mass spectrometry to detect protein interactions,eliminating the interference caused by mass spectrometry detection.Because in most quantitative studies of protein and metal ion interactions,we usually add all binding ratio of charged states,and then calculate binding constant or affinity data;or take the most abundant one charge state to calculate binding constant.However,if charge state has an effect on protein-metal ion specific interactions,these calculations may both have problems.In addition,we also conducted an in-depth study of the effect of charge state on nonspecific interactions generated in ESI spray process.Mathematical model calculations were used here to distinguish specific binding and nonspecific binding in traditional ESI-MS spectra,and to study the relationship between nonspecific binding and charge states separately.Different nonspecific binding systems were selected to systematically study the mechanism of charge state effects.Finally,inductive electrospray(IESI)was used as ion source to generate millisecond microelectrophoresis before spraying.In the microelectrophoresis,excess metal ions and protein complexes were separated,effectively eliminating nonspecific binding problems existing in traditional ESI-MS.A more authentic protein-metal ion binding states were obtained.