Development And Application Of "In-Cell” Protein Mass Spectrometry

Conventional protein mass spectrometry relies on other techniques,including high-performance liquid chromatography and gel electrophoresis,to separate and purify the proteins in the complex systems and environment.On the one hand,these time-consuming purification processes seriously change the native environment,resulting in mass spectrometric detection of proteins that are not necessarily with the specific functions in cells.On the other hand,the sample preparation processes may directly result in the loss of low abundance proteins,which may play an indispensable role in some diseases and even cancer developing processes.And this is very dangerous,because a lot of clinical diagnosis and medical evaluations are based on the offline results.If the results are not reliable,it may lead the doctor to make infavorable judgments for disease diagnosis and treatment protocols.In summary,the development of "in-cell" protein mass spectrometry,which can directly identify and characterize intracellular proteins,is important for both the fundamental research and biomedical application.This PhD project is aimed at developing the "in-cell" protein mass spectrometry,which is a new concept,and the goal is to reveal the true intracellular protein structures and protein interactions.Technically,the core is using millisecond micro-electrophoresis to remove the matrix from complex mixtures,and we also used some additional methods include native MS and ion mobility mass spectrometry.For application,it can provide a reliable basis for protein science,and also may appeal some industrial applications including anticancer drug screening at the cellular level.This dissertation is divided into three parts as follows:1.Instrumental development for "in-cell" protein mass spectrometry platformThe first part introduces how to integrate continuous ultrafast micro-electrophoresis into nanospray.To achieve ultrafast millisecond micro-electrophoretic separation:a)to build a single pulsed DC high voltage power supplier,micro electrophoresis around 1 Hz(within a single electrophoresis duration,200 ms),b)to build a continuous controlled pulse power supplier.Micro-electrophoresis behavior of several Hz to several thousand Hz can be continuously adjusted.Through rapid electrophoresis,preliminary separation of matrixes and target compounds in a complex mixtures was achieved,so as to reduce the the matrix effects in subsequent nanospray processes.We used high concentrated salt systems to mimic the complex matrixes,and then to evaluate its separation efficiency and explore its separation mechanism.We also designed experiments on capillary electrophoresis electrospray mass spectrometry interface,mitigation strategies for the alleviation of ion suppression effect in conventional spray ionization and nanoelectrospray ionization.2."In-cell" protein mass spectrometry for protein analysisThis part mainly introduces how to use the "In-cell" protein mass spectrometry techniques for protein structural characterization and functional interrogation:protein identification in pure solution and in complex system.In pure solution,we studied the protein structures,including sequence coverage of disulfide proteins;stepwise unfolding of single domain proteins;combining "in-cell" protein mass spectrometry with ion mobility spectrometry,taking hemoglobin as a model,we also study protein disassembly pathway and quaternary structures.For the complex systems,we showed how to use the "in-cell" protein mass spectrometry to qualitatively and quantitatively identify proteins in high-salt conditions,in fetal bovine serum,in cell lysates and from whole living cells.3."In-cell" protein mass spectrometry for protein interaction analysisIn this section we will introduce the application of "in-cell" protein mass spectrometry in protein interaction analysis,which contains protein-metal interaction and protein-protein interactions.The main model proteins include zinc finger protein(NCp7,Sp1)and calmodulin(calmodulin,CaM).We also tested the availability of "in-cell" protein mass spectrometry both in pure solution and whole living cells for protein interaction analysis.This approach avoids problems resulting from the complicated cellular environment.In this manner we demonstrate the rapid identification of intact proteins from living E.coli cells including the complexation of calmodulin with calcium ion.The latter showed different binding states from those observed in in-vitro studies.These observations also reveal in-vitro measurements do not necessarily represent the actual situation in living cells,which could also be one of the reason for the loss of efficacy of some anticancer drugs in cells or in tumor environment though the in-vitro results are considerably effective.