Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.     

Charge ratio analysis method: approach for the deconvolution of electrospray mass spectra

A new method to interpret electrospray mass spectral data based on calculating the ratio of mass-to-charge (m/z) values of multiply charged ions is described. The mass-to-charge ratios of any two multiply charged ions corresponding to a single compound are unique numbers that enable the charge states for each ion to be unequivocally identified. The multiply charged ions in electrospray mass spectra originate from the addition or abstraction of protons, cations, or anions to and from a compound under analysis. In contrast to existing deconvolution processes, the charge ratio analysis method (CRAM), identifies the charge states of multiply charged ions without any prior knowledge of the nature of the charge-carrying species. In the case of high-resolution electrospray mass spectral data, in which multiply charged ions are resolved to their isotopic components, the CRAM is capable of correlating the isotope peaks of different multiply charged ions that share the same isotopic composition. This relative ratio method is illustrated here for electrospray mass spectral data of lysozyme and oxidized ubiquitin recorded at low- to high-mass resolution on quadrupole ion trap and Fourier transform ion cyclotron mass spectrometers, and theoretical data for the protein calmodulin based upon a reported spectrum recorded on the latter.     

Effect of experimental parameters on the ESI FT-ICR mass spectrum of fulvic acid

Fulvic acid (FA) is a heterogeneous mixture of organic macromolecules found in the waters, soils, and sediments of the earth's surface. The ability of electrospray ionization (ESI) to effectively transfer large ions from the solution phase to the gas phase and the coupling of ESI to the high-mass-resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provide a potential method for the mass spectrometric analysis of FA. Positive- and negative-ion ESI FT-ICR MS analyses of four reference International Humic Substances Society FAs were performed. The spray solution composition was found to have a dramatic effect on the ion distributions, with high-mass aggregates (m/z approximately 2000-4000) being formed in less polar spray solutions. Positive-ion spectra for each FA obtained under optimum conditions resulted in number-average molecular weights ranging from 1700 to 1900. The mass spectra were extremely complex, with ion distributions on the order of m/z approximately 500-3000. The presence of more than one ion at each nominal mass was routinely observed. Negative-ion ESI analysis of the FA samples resulted in the observation of multiply charged ions whose distributions could be affected by the acidification of the spray solution. Solution parameters which have been reported to affect molecular weight distributions of FA such as pH, ionic strength, and concentration of multivalent cations were found to have little or no effect on the observed m/z distributions.     

Dynamics of iron release from transferrin N-lobe studied by electrospray ionization mass spectrometry

Transferrins constitute a class of metalloproteins that are involved in circulatory iron transport in a variety of species. The metal ion-binding properties of these proteins have been the focus of extensive research efforts in the past decade due to their extreme importance in a variety of biological and healthcare-related fields. The large size of these proteins, as well as the presence of high-spin metal ions (e.g., Fe3+), limits the use of NMR. In this work, we report on the use of electrospray ionization mass spectrometry (ESI MS) to study dynamics of the transferrin system in vitro under conditions that are designed to mimic the endosomal environment. ESI MS is shown to provide valuable insights into the mechanistic aspects of metal ion-binding/release by transferrins and is complementary to other spectroscopic techniques. Conformational stability of the complex is evaluated based on the appearance of the charge-state distribution of protein ions, while the composition of the protein-ligand complex is determined based on the mass of the protein ions. In the absence of iron chelators, a stepwise dissociation of the ternary complex (protein-metal ion-synergistic anion) is observed as the solution pH is gradually decreased. Although the release of synergistic anion from the complex is initiated at typical endosomal pH levels (i.e., 5.5), metal ion remains largely bound to the protein until the pH is lowered to a level of approximately 4.5. Under these conditions, a significant fraction of the protein populates unfolded conformations. In stark contrast to this behavior, addition of an iron chelating agent (citrate) to the protein solution results in facile iron release at typical endosomal pH levels without any detectable unfolding of the protein. The mass spectral data lends further credibility to the notion that the holoprotein samples conformations that are specific to the apo form (e.g., "open conformation"), from which iron dissociation most likely occurs. The results of the present study demonstrate that ESI MS can be used to model metal ion release from transferrin under conditions that are designed to mimic the physiological environment.     

Ionization and fragmentation of humic substances in electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) was combined with ultra-high-resolution Fourier transform-ion cyclotron resonance mass spectrometry (FTICR MS) to characterize complex humic and fulvic acid mixtures. Lower than expected molecular weight distributions previously observed for humics when analyzed by ESI-MS have fueled speculation about a bias in favor of low molecular weight. Multiply charged ions, ionization suppression, and sample fragmentation have all been suggested as sources of this low molecular weight bias. In this work, resolution of the individual components of humic mixtures within a 1 mass-to-charge unit window was accomplished by FTICR MS at 9.4 T. At mass resolving powers between 60,000 (high mass) and 120,000 (low mass), it was possible to determine that virtually all ions present in spectra of Suwannee River fulvic and humic acid are singly charged, thus eliminating inadequate accounting for multiply charged ions as a primary source of any low molecular weight bias. The high-resolution mass spectra also revealed the presence of molecular families containing ions that differ from each other in degree of saturation, functional group substitution (primarily CH vs N and CH4 vs O), and number of CH2 groups. Ionization suppression and ion fragmentation were addressed for humic and fulvic acid mixtures and well-characterized poly(ethylene glycol) (PEG) mixtures with average molecular weights of 8000 and 10,000. Although these high molecular weight PEG mixtures fragment extensively under traditional positive-ion mode ESI conditions, similar fragmentation could not be confirmed for humic and fulvic acid mixtures.     

Gas-phase interference-free analysis of protein ion charge-state distributions: detection of small-scale conformational transitions accompanying pepsin inactivation

Analysis of protein ion charge-state distributions in electrospray ionization (ESI) mass spectra has become an indispensable tool in the studies of protein dynamics. However, applications of this technique have been thus far limited to detection of large-scale conformational transitions, which typically change the extent of multiple charging in a very significant way. However, more subtle conformational changes often elude detection, since the resulting changes of the extent of multiple charging are often smaller than the charge-state shifts caused by other external factors. Proton-transfer reactions involving protein ions and residual solvent molecules are the major extrinsic factors causing changes of charge-state distributions unrelated to conformational transitions. Since the extent of such reactions depends on the amount of various solvent components transferred to the ESI interface, profound changes of solvent composition may affect protein ion charge-state distributions not only by affecting protein higher order structure in solution but also through modulation of the efficiency of proton-transfer reactions in the gas phase. Here we demonstrate that it is possible to choose experimental conditions in such a way that the influence of gas-phase ion chemistry on protein ion charge-state distributions is not altered over a wide pH range. This methodology (gas-phase interference-free analysis of protein ion charge-state distributions, or GIFPICS) is sensitive enough to allow detection of pepsin inactivation under mildly acidic conditions. Pepsin is active and tightly folded in its native strongly acidic environment. Inactivation of pepsin at mildly acidic pH is not accompanied by global unfolding, as spectroscopic measurements suggest the protein remains compact. GIFPICS provides a means to observe this small-scale conformational transition that does not result in protein unfolding and may in fact elude detection by traditional spectroscopic techniques.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Electrothermal supercharging of proteins in native electrospray ionization

The formation of high charge-state protein ions with nanoelectrospray ionization (nESI) from purely aqueous ammonium bicarbonate solutions at neutral pH, where the proteins have native or native-like conformations prior to ESI droplet formation, is demonstrated. This "electrothermal" supercharging method depends on the temperature of the instrument entrance capillary, the nESI spray potential, and the solution ionic strength and buffer, although other factors almost certainly contribute. Mass spectra obtained with electrothermal supercharging appear similar to those obtained from denaturing solutions where charging beyond the total number of basic sites can be achieved. For example, a 17+ ion of bovine ubiquitin was formed by nESI of a 100 mM ammonium bicarbonate, pH 7.0, solution, which is three more charges than the total number of basic amino acids plus the N-terminus. Heating of the ESI droplets in the vacuum/atmosphere interface and the concomitant denaturation of the protein in the ESI droplets prior to ion formation appears to be the primary origin of the very high charge-state ions formed from these purely aqueous, buffered solutions. nESI mass spectra resembling those obtained under traditional native or denaturing conditions can be reversibly obtained simply by toggling the spray voltage between low and high values.     

Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization.

Serum albumin proteins, Mr approximately 66 kDa, from 10 different species (bovine, human, rat, horse, sheep, goat, rabbit, dog, porcine, and guinea pig) have been studied by electrospray ionization mass spectrometry (ESI-MS) and tandem MS using a triple-quadrupole instrument. The effectiveness of collisional activation for the multiply charged albumin ions greatly exceeds that for singly charged ions, allowing an extension by a factor of at least 20 to the molecular mass range for obtaining sequence-specific product ions by tandem MS. Efficient dissociation is largely attributed to "preheating" in the interface Coulombic instability and the large number of collisions. Increasing the electric field in the intermediate pressure region, between the nozzle-skimmer elements of the atmospheric pressure/vacuum interface, allows fragmentation of the multiply protonated (to 96+) molecules produced by ESI. The most abundant dissociation product ions assigned have a low charge state (2+ to 5+) and are attributed to "bn" mode species from cleavage of the -CO-N- peptide backbone bonds. Particularly abundant dissociation products originate from regions near residues n = 20-25 from the NH2 terminus for parent ions of moderate charge (approximately 50+). Collisionally activated dissociation (CAD) mass spectra from porcine serum albumin, in contrast to the other albumins, also gave prominent singly charged "yn" fragments formed from cleavages near the COOH terminus. Tandem mass spectrometry (MS/MS) of the multiply charged molecular ions, and of fragment species produced by dissociation in the interface (i.e., effective MS/MS/MS), produced similar "bn" species and served to confirm spectral assignments. We also show that ESI mass spectra allow a qualitative assessment of protein microheterogeneity and, in some cases, resolution of major contributions. The physical and analytical implications of the results are discussed, including the identification of possible errors in previously published sequences.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Monitoring and modeling of protein processes using mass spectrometry, circular dichroism, and multivariate curve resolution methods

Mass spectrometry has recently become one of the major analytical tools to study biomolecular structure and function. Ionization techniques, such as electrospray ionization (ESI), desorb biomolecules from solution to the gas phase keeping practically intact their natural structure. ESI applied to a protein solution produces a mixture of multiply charged ions, the ion charge distribution of which depends on the oligomeric form (mass) and on the protein surface exposed (amount of accommodated charges) of the related protein conformation. ESI-MS provides an efficient way to monitor protein processes; however, the ionic contributions of the different protein conformations involved usually overlap, and the use of chemometric tools is necessary to unravel the information related to the pure conformations that the biomolecule adopts along the process. Multivariate curve resolution-alternating least squares applied to MS-monitored protein processes provides the concentration profiles associated with the different protein conformations occurring during the process and the related pure mass spectra. The concentration profiles, in this context, the ionic contributions, describe the process mechanism and the structural information derived from the pure mass spectra characterizes the involved conformations. Mass spectra can be expressed schematically through percentages of base peak intensity. This chemical transformation compresses significantly the raw spectra and allows for an easier application of natural MS-related constraints, such as the presence of only one maximum, i.e., the base peak of a particular conformation, into the resolution of the pure signals. The combination of mass spectrometry and multivariate curve resolution methods is used to elucidate the mechanism of the pH-induced conformation changes of the bovine beta-lactoglobulin. As a final step, MS data are fused with circular dichroism data and are simultaneously analyzed to ensure and confirm that all the previously detected MS conformations really exist in solution and are an artifact of neither the ionization process nor their chemometric resolution.     

Infrared multiphoton dissociation in an external ion reservoir.

In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 microns from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-to-noise ratio and fragment yield, particularly for larger, more highly charged fragment ions.     

Generation of highly charged peptide and protein ions by atmospheric pressure matrix-assisted infrared laser desorption/ionization ion trap mass spectrometry

We show that highly charged ions can be generated if a pulsed infrared laser and a glycerol matrix are employed for atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry with a quadrupole ion trap. Already for small peptides like bradykinin, doubly protonated ions form the most abundant analyte signal in the mass spectra. The center of the charge-state distribution increases with the size of the analyte. For example, insulin is detected with a most abundant ion signal corresponding to a charge state of four, whereas for cytochrome c, the 10 times protonated ion species produces the most intense signal. Myoglobin is observed with up to 13 charges. The high m/z ratios allow us to use the Paul trap for the detection of MALDI-generated protein ions that are, owing to their high molecular weight, not amenable in their singly protonated charge state. Formation of multiple charges critically depends on the addition of diluted acid to the analyte-matrix solution. Tandem mass spectra generated by collision-induced dissociation of doubly charged peptides are also presented. The findings allow speculations about the involvement of electrospray ionization processes in these MALDI experiments.     

Electrospray ionization mass spectrometry of tetracycline, oxytetracycline, chlorotetracycline, minocycline, and methacycline

The effects of mobile-phase additives and analyte concentration on electrospray ionization mass spectra of a series of tetracyclines were investigated in both positive and negative ion modes. Only [M + H](+) and [M - H](-) ions were observed. The greatest sensitivity as [M + H](+) ions was obtained with 1% acetic acid and the greatest sensitivity as [M - H](-) ions was obtained using 50 mM ammonium hydroxide. Sensitivities in the positive ion mode were greater than those in the negative ion mode. The sensitivity as [M + H](+) showed no systematic variation with pH; however, the sensitivity as [M - H](-) did increase with increasing pH. A larger linear range was observed for [M - H](-) than for [M + H](+) ions. Both [M + Na](+) and [M + H](+) ions were observed with 0.5 mM sodium acetate and sodium iodide, but no adduct ions were observed with ammonium acetate. Some M(2)H(+) ions were observed at higher concentrations. Cluster ions, Na(NaOAc)(n)(+) or Na(NaI)(n)(+), but no sample ions were observed using 5 mM salts. The data suggest that mechanisms in addition to solution ionization are involved in the formation of the ESI sample ions. The utility of mobile phases containing 1% HOAc or 50 mM NH(4)OH was demonstrated for chromatographic separations.     

Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer

A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.     

Problems on computer processing of SIMS spectra

The main problems of the interpretation of low resolution secondary ion mass spectra are treated and an evaluation program is presented for SIMS spectra. The programme searches automatically for monoatomic single and double charged ions as well as isoatomic and heteroatomic single charged cluster ions which are the most typical for SIMS spectra and provides also the total ionic current of these species.     

A capillary mixer with adjustable reaction chamber volume for millisecond time-resolved studies by electrospray mass spectrometry

A novel continuous-flow apparatus for on-line kinetic studies of (bio)chemical solution-phase processes by electrospray ionization mass spectrometry (ESI-MS) is described. The device is based on two concentric capillaries. Fluid is released from the inner capillary into the intercapillary space, where it mixes with solution flowing through the outer capillary, thus initiating the reaction of interest. Gas-phase analyte ions are formed near the tip of the outer capillary by pneumatically assisted ESI. This setup allows the mixer to be placed directly within the ion source, thus providing a minimal dead volume of ~8 nL. Time-resolved data can be recorded in both "spectral" and "kinetic" modes. In the former case, the position of the inner capillary is fixed at various points, such that entire mass spectra can be recorded for selected reaction times. For experiments in kinetic mode, the mass spectrometer monitors the signal intensity at selected m/z values, while the inner capillary is continuously pulled back, thus providing intensity-time profiles for specific reactive species. A theoretical framework is developed that allows the measured kinetics to be analyzed by taking into account the effects of laminar flow within the reaction capillary. Failure to take these effects into account results in erroneous rate constants. Studies on the demetalation kinetics of chlorophyll reveal that the apparatus can reliably measure rate constants up to at least 100 s-1. This represents a substantial improvement over previous ESI-MS-based kinetic methods. Spectral mode experiments on the refolding of ubiquitin show the changing proportions of denatured and tightly folded protein subpopulations in solution. When monitored in kinetic mode, the refolding process was found to proceed with a rate constant of 5.2 s-1.     

Mapping of protein disulfide bonds using negative ion fragmentation with a broadband precursor selection

Fast mapping of disulfide bonds in proteins containing multiple cysteine residues is often required in order to assess the integrity of the tertiary structure of proteins prone to degradation and misfolding or to detect distinct intermediate states generated in the course of oxidative folding. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures utilizes the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode. Cleavages of cysteine side chains result in efficient dissociation of disulfide bonds and produce characteristic signatures in the fragment ion mass spectra. While cleavages of other side chains result in insignificant loss of mass and full retention of the peptide ion charge, dissociation of external disulfide bonds results in physical separation of two peptides and, therefore, significant changes of both mass and charge of fragment ions relative to the precursor ion. This feature allows the fragment ions generated by dissociation of external disulfide bonds to be easily detected and identified even if multiple precursor ions are activated simultaneously. Such broadband selection of precursor ions for consecutive activation is achieved by lowering the dc/rf amplitude ratio in the first quadrupole filter of a hybrid quadrupole time-of-flight mass spectrometer. The feasibility of the new method is demonstrated by partial mapping of disulfide bridges within a 37-kDa protein containing 16 cysteine residues and complete disulfide mapping within a lysozyme (14.5 kDa) containing 8 cysteine residues. In addition to detecting peptide pairs connected by a single external disulfide, the new method is also shown to be capable of identifying peptides containing both external and internal disulfide bonds. The two major factors determining the efficiency of disulfide mapping using the new methodology are the effectiveness of proteolysis and the ability of the resulting proteolytic fragments to form multiply charged negative ions.     

Selective ion filtering by digital thresholding: a method to unwind complex ESI-mass spectra and eliminate signals from low molecular weight chemical noise

In this work, we present a simple method by which to preferentially detect either high molecular weight or low molecular weight ions generated by electrospray ionization. This approach, termed selective ion filtering by digital thresholding (SIFdT) is demonstrated on a commercial ESI-TOF instrument that employs a fast digitizer coupled to a microchannel plate detector. The digital representation of each individual scan is digitally filtered prior to spectral coaddition. As larger, more highly charged ions induce a more intense response than low molecular weight singly charged species, a digital threshold can be set that precludes the detection of singly charged species yet permits the efficient detection of larger, more highly charged species. In this work, we demonstrate the applicability of this approach to eliminate low molecular weight chemical noise in ESI-TOF spectra of oligonucleotide and protein ions, demonstrate improved dynamic range for analyte solutions containing high levels of low MW constituents, and show that spectra acquired at different digital thresholds can be subtracted to yield spectra of low molecular weight constituents with improved mass measurement accuracies. A notional scheme is presented in which an alternative digitization approach is employed using multiple differentially thresholded data streams to allow improved internal mass calibration and higher resolution ion partitioning.     

Structural analysis of ionic organotin(IV) compounds using electrospray tandem mass spectrometry

A novel electrospray ionization (ESI) mass spectrometric approach for the structure elucidation of ionic organotin(IV) compounds or complexes with weakly bonded ligands as for example monodentate carboxylates or sulfonates is proposed using both positive-ion and negative-ion ESI tandem mass spectra. The ionization mechanism of organotin(IV) compounds involves the cleavage of the most labile bond with an ionic character yielding two complementary ions, [Cat]+ and [An]-. Positively charged species containing tin atom, [Cat]+, are analyzed in the positive-ion mode and negatively charged species without the tin atom, [An]-, in the negative-ion mode. Fragmentation patterns of [C24H29N2Sn]+, [C21H22NSn]+, and [C17H30NSn]+ ions are proposed based on the detailed interpretation of MSn spectra, which is simplified by an easy recognition of characteristic tin isotopic clusters in particular fragment ions. Proposed fragmentation mechanisms are supported by comparison with MSn spectra of deuterium-labeled analogues. The applicability of this method is illustrated on two sets of organotin(IV) compounds, including seven [2,6-bis(dimethylaminomethyl)phenyl]diphenyltin(IV) derivatives with small inorganic counteranions X (Br, NO3, SCN, BF4, SeCN, CN, PF6), six organotin(IV) complexes containing two C,N-chelating ligands with azo dyes, and the identification of unknown hydrolysis products.