Regulation Of Ionization Behavior In ESI-MS And Its Application

Mass spectrometry(MS)is a sensitive tool for simultaneous multiple components analysis,it can also provide structural information when tandem mass spectrometry is used.Electrospray ionization(ESI)can transform polar molecules in the solution into gas-phase ions.ESI is powerful method for the ionization of proteins,metabolites,lipids and nucleic acids,etc.,which makes it a widely used ion source in MS.The most challenging part in ESI-MS lies in the ionization of a target molecule when complex matrix is also presented in the solution.To alleviating the adverse effects of matrix on the ionization of a target molecule,it is therefore necessary to perform pretreatment like separation or enrichment prior to a mass spectrometric analysis.Due to the limited sample amount,most of the current pretreatment methods are not applicable for ultra-small single-cell samples.Moreover,gentle desolvation conditions and buffers containing salts are needed in native mass spectrometry to maintain the native-like conformers of proteins and the binding states of complexes in the transition from solution to gas phase.In this thesis,we demonstrated the use of various regulating methods to enhance the ionization of target molecules for single-cell analysis and native mass spectrometric analysis.A gas-phase synthetic method for phosphorylation of ribose and ribonucleosides was also demonstrated.The main work is as following:1.Synchronized polarization induced electrospray ionization and its application in single cell analysisIn ESI,basic molecules are preferred to form positively charged ions and acidic molecules are preferred to form negatively charged ions.The molecule diversity in the single-cell samples demands that both positive and negative polarity of the mass analyzer should be used for a comprehensive profiling.A new ionization source called synchronized polarization induced electrospray(SPI-ESI)was developed,which is capable of simultaneously obtaining both positive-ion and negative-ion mass spectra through one measurement.In SPI-ESI,periodic altermating current square wave voltage is applied to induce the bipolar spray and both positive-ion and negative-ion mass spectra are obtained by synchronizing the mode of mass analyzer with the bipolar spray process.We have successfully applied SPI-ESI to the analysis of single-cell samples.By combining both positive-ion and negative-ion mass spectra,a comprehensive profiling of metabolites and lipid in single cells is achieved.Ultimately,86 and 94 components are identified from single Allium cepa cells and single PC-12 cells.Moreover,ultralow spray flow rate(?30 pL/min)is achieved in SPI-ESI without loss of its sensitivity.The decrease of flow rate prolongs the MS signal duration from single-cell samples to acquire ms2 data for components determination.2.Alleviation of nonspecific metal adduction in electrospray ionization mass spectrometry by using nanoemittersESI-MS is a powerful tool to identify peptides and proteins.However,the performance of ESI-MS can be susceptible because of the ion suppression effect,especially when some involatile salts(even at trace amounts)are also present in the samples.The signal intensity of the peptides or proteins can be reduced due to the formation of metal adducts,which also depress the sensitivity of ESI-MS.To avoid the adverse effects of involatile salts on detection of peptides or proteins in ESI-MS,a nano-sized glass emitter was used to perform electrospraying.In comparison with ESI using a microemitter(e.g.a 1.2 ?m opening),ESI from a nanoemitter(e.g.a 120 nm opening)is characterized by alleviating salt cluster ions and nonspecific metal adduction to peptide or protein ions.Smaller initial charged droplets generated from a nanoemitter contain less salt and need less fission steps to release the gas-phase ions.Thus,the enrichment effect of salt was not as significant as that of a microemitter,resulting in the disappearing of salt cluster peaks in high mass-to-charge(m/z)region.The pH decrease of the aqueous solution inside a nanoemitter by the exclusion-enrichment effect also contributes to regulating the molecular ions into protonated ones.The use of a nanoemitter can improve the matrix tolerance and no pretreatment such as desalting is needed even in the presence of salt at millimolar concentration,making it a useful tool for determining the binding states of protein complexes.3.Nonspecific metal adduction as a signature to gauge the zwitterionic interactions of proteinsThe structural characterization of proteins and their interaction network mapping in the gas phase using mass spectrometry highlight the need to preserve their most native?like conformers when proteins are transferred from solution to gas phase.zwitterionic interactions are weak bonds between oppositely charged residues in proteins,which make essential contributions to the stabilization of compact conformers of many proteins in both the gas and condensed phase.It?s still unclear whether the zwitterionic states of the protein found in solution can be retained or not when desolvated and very rare studies have to date investigated the desolvation condition that can reliably maintain the zwitterionic interactions.Using the nonspecific Na+ adduction as a novel signature,we show that the zwitterionic states of proteins can be preserved when a moderated droplet desolvation condition(e.g.temperature<30? and drying gas flow rate=0.8 L/min)is used in native electrospray ionization mass spectrometry.The very low-level nonspecific metal adduction to proteins under such conditions also enables rapid and direct determination of the binding states of metal-binding proteins and sensitive detection of proteins from solutions containing highly concentrated involatile salts(e.g.,50 mM NaCl).4.Regioselective 5'-position phosphorylation of ribose and ribonucleosides in the activated pyrophosphate complex in the gas phasePhosphorylation is an essential chemical reaction in all known living systems and it generates phosphorylated biomolecules.Abiotic production of phosphorylated biomolecules,especially nucleotides,has also attracted widespread interest as it is relevant to the origin of life.We present a rapid,efficient and regioselective phosphorylation method at the 5'-position of unprotected ribose and ribonucleosides with pyrophosphate in the gas phase,which involves the formation of anionic complexes via electrospray ionization and collisional activation to induce phosphorylation within the complexes.Under the optimal collision energy,13.2%of the anionic complex between ribose and pyrophosphate can be converted to ribose 5-phosphate.In the case of ribonucleosides,the full set of the four canonical ribonucleotides(namely adenosine 5-phosphate,uridine 5-phosphate,guanosine 5-phosphate and cytidine 5-phosphate)can be successfully prepared with a conversion rate of 18.9%,29.6%,17.6%and 11.3%,respectively.This work also suggested that collisional activation might have played a role in promoting the chemical reaction occurred in the ESI process.