Multiply charged ions in ionspray tandem mass spectrometry

Multiply charged anions of some 30 species containing multiple carboxylic, sulphonic and/or phosphoric groups generated by ionspray were studied by using tandem mass spectrometry (MS/MS). Two trends emerged: (1) lower-charged ions are preferentially evaporated, and (2) for more highly charged ions, evaporability is a function of ion size. Triply and quadruply charged ions were observed for azo dyes with molecular masses in the 700-900 Dalton range. Daughter-ion mass spectra of selected multiply charged ions are presented.     

Enhancement of mass spectrometry performance for proteomic analyses using highfield asymmetric waveform ion mobility spectrometry (FAIMS)

Remarkable advances in mass spectrometry sensitivity and resolution have been accomplished over the past two decades to enhance the depth and coverage of proteome analyses. As these technological developments expanded the detection capability of mass spectrometers, they also revealed an increasing complexity of low abundance peptides, solvent clusters and sample contaminants that can confound protein identification. Separation techniques that are complementary and can be used in combination with liquid chromatography are often sought to improve mass spectrometry sensitivity for proteomics applications. In this context, high‐field asymmetric waveform ion mobility spectrometry (FAIMS), a form of ion mobility that exploits ion separation at low and high electric fields, has shown significant advantages by focusing and separating multiply charged peptide ions from singly charged interferences. This paper examines the analytical benefits of FAIMS in proteomics to separate co‐eluting peptide isomers and to enhance peptide detection and quantitative measurements of protein digests via native peptides (label‐free) or isotopically labeled peptides from metabolic labeling or chemical tagging experiments. Copyright © 2015 John Wiley & Sons, Ltd.     

Matrix Assisted Ionization: New Aromatic and Nonaromatic Matrix Compounds Producing Multiply Charged Lipid, Peptide, and Protein Ions in the Positive and Negative Mode Observed Directly from Surfaces

Matrix assisted inlet ionization (MAII) is a method in which a matrix:analyte mixture produces mass spectra nearly identical to electrospray ionization without the application of a voltage or the use of a laser as is required in laserspray ionization (LSI), a subset of MAII. In MAII, the sample is introduced by, for example, tapping particles of dried matrix:analyte into the inlet of the mass spectrometer and, therefore, permits the study of conditions pertinent to the formation of multiply charged ions without the need of absorption at a laser wavelength. Crucial for the production of highly charged ions are desolvation conditions to remove matrix molecules from charged matrix:analyte clusters. Important factors affecting desolvation include heat, vacuum, collisions with gases and surfaces, and even radio frequency fields. Other parameters affecting multiply charged ion production is sample preparation, including pH and solvent composition. Here, findings from over 100 compounds found to produce multiply charged analyte ions using MAII with the inlet tube set at 450?°C are presented. Of the compounds tested, many have ?COH or ?CNH₂ functionality, but several have neither (e.g., anthracene), nor aromaticity or conjugation. Binary matrices are shown to be applicable for LSI and solvent-free sample preparation can be applied to solubility restricted compounds, and matrix compounds too volatile to allow drying from common solvents. Our findings suggest that the physical properties of the matrix such as its morphology after evaporation of the solvent, its propensity to evaporate/sublime, and its acidity are more important than its structure and functional groups.     

Tandem mass spectrometry of half-generation PAMAM dendrimer anions

Ions derived from negative electrospray ionization of polyamidoamine (PAMAM) dendrimer generation 0.5 were subjected to ion trap tandem mass spectrometry. Ion/ion proton transfer reactions were used to manipulate the charge states of PAMAM precursor ions to form lower charge states from those initially formed by electrospray, as well as to facilitate the interpretation of the product ion mass spectra. Most of the products derived from dendrimer precursor ions could be rationalized by retro-Michael decomposition reactions. The dominant fragmentation channels are highly dependent on the composition of the counter-ions, which in this case are restricted to different numbers of sodium ions and protons, and whether the precursor ion is multiply charged or singly charged. An interpretation is given that is consistent with all of the observations made with the various anions associated with this study. The nature of the structural information that can be obtained via ion trap tandem mass spectrometry of the dendrimers is dependent on the types of precursor ions subjected to study. The tandem mass spectrometry data also provided information about the structure of faulty synthesis products present in the PAMAM dendrimer sample.     

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.     

Tandem mass spectra of tryptic peptides at signal-to-background ratios approaching unity using electrospray ionization high-field asymmetric waveform ion mobility spectrometry/hybrid quadrupole time-of-flight mass spectrometry

High-field asymmetric waveform ion mobility spectrometry (FAIMS) has been coupled to a quadrupole time-of-flight mass spectrometer for the tandem mass spectrometric analysis of tryptic peptides of pig hemoglobin. Using FAIMS, low levels (fmol/microL) of multiply charged tryptic peptides were separated from relatively intense chemical background such that their tandem mass spectra (MS/MS) lacked many background-related fragment ions observed using a conventional ESI-QqTOFMS instrument. Substantial improvements in both first-order and tandem mass spectra were realized while maintaining approximately the same absolute intensities.     

Laserspray ionization using an atmospheric solids analysis probe for sample introduction

A newly introduced high sensitivity laserspray (LSI) mass spectrometry (MS) method that uses laser ablation of a matrix/analyte mixture at atmospheric pressure (AP) to obtain multiply charged ions from nonvolatile as well as high-mass compounds is now implemented using a simple probe device. The probe used in the LSI approach was originally designed for sample introduction into an AP ionization source using the atmospheric solids analysis probe (ASAP) method. Multiply charged mass spectra of peptides and proteins in 2,5-dihydroxybenzoic acid matrix were readily obtained on two mass spectrometers from different manufacturers with sample introduction using melting point tubes. Here we demonstrate rapid analysis by placing four peptide and protein samples on a single melting point tube. Mass spectra were obtained at high-resolution and using ion mobility spectrometry/MS.     

Evolution of instrumentation for the study of gas-phase ion/ion chemistry via mass spectrometry

The scope of gas-phase ion/ion chemistry accessible to mass spectrometry is largely defined by the available tools. Due to the development of novel instrumentation, a wide range of reaction phenomenologies has been noted, many of which have been studied extensively and exploited for analytical applications. This perspective presents the development of mass spectrometry-based instrumentation for the study of the gas-phase ion/ion chemistry in which at least one of the reactants is multiply charged. The instrument evolution is presented within the context of three essential elements required for any ion/ion reaction study: the ionization source(s), the reaction vessel or environment, and the mass analyzer. Ionization source arrangements have included source combinations that allow for reactions between multiply charged ions of one polarity and singly charged ions of opposite polarity, arrangements that enable the study of reactions of multiply charged ions of opposite polarity and, most recently, arrangements that allow for ion formation from more than two ion sources. Gas-phase ion/ion reaction studies have been performed at near atmospheric pressure in flow reactor designs and within electrodynamic ion traps operated in the mTorr range. With ion trap as a reaction vessel, ionization and reaction processes can be independently optimized and ion/ion reactions can be implemented within the context of MSn experiments. Spatial separation of the reaction vessel from the mass analyzer allows for the use of any form of mass analysis in conjunction with ion/ion reactions. Time-of-flight mass analysis, for example, has provided significant improvements in mass analysis figures of merit relative to mass filters and ion traps.     

Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry

A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed.     

Charge Transfer and Metastable Ion Dissociation of Multiply Charged Molecular Cations Observed by Using Reflectron Time-of-Flight Mass Spectrometry

A multiply charged molecule expands the range of a mass window and is utilized as a precursor to provide rich sequence coverage; however, reflectron time-of-flight mass spectrometer has not been well applied to the product ion analysis of multiply charged precursor ions. Here, we demonstrate that the range of the mass-to-charge ratio of measurable product ions is limited in the cases of multiply charged precursor ions. We choose C₆F₆ as a model molecule to investigate the reactions of multiply charged molecular cations formed in intense femtosecond laser fields. Measurements of the time-of-flight spectrum of C₆F₆ by changing the potential applied to the reflectron, combined with simulation of the ion trajectory, can identify the species detected behind the reflectron as the neutral species and/or ions formed by the collisional charge transfer. Moreover, the metastable ion dissociations of doubly and triply charged C₆F₆ are identified. The detection of product ions in this manner can diminish interference by the precursor ion. Moreover, it does not need precursor ion separation before product ion analysis. These advantages would expand the capability of mass spectrometry to obtain information about metastable ion dissociation of multiply charged species.     

Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

Mechanism of production of ions in electrospray mass spectrometry

A mechanism for the production of multiply charged molecular ion species in electrospray mass spectrometry (ES-MS) is still required. A concise discussion of a recently published ionic solution equilibrium model offering a partial mechanism is presented. That publication proposed that the ion abundance–charge profile could be fitted by one or a series of superimposed Gaussian functions, in accord with a solution equilibrium model. It is shown that indeed a simulated mass spectrum based on a solution odel can compare well with the observed spectrum. However, some new and recently published experimental evidence is presented which shows that the ES mass spectra of many proteins give rise to multiply charged molecular ions which carry higher charges than those calculated by the model. Further, the ion abundance–charge profile is very sensitive to some experimental parameters, e.g. cone voltage; it does not necessarily reflect the solution or gaseous ion populations in the mass spectrometer source. Therefore, the concept that gaseous multi-charged ions originate from equivalently charged solvated ions in electrically neutral solution must be treated with caution.     

Tandem mobility mass spectrometry study of electrosprayed tetraheptyl ammonium bromide clusters

Multiply charged electrospray ions from concentrated solutions of Heptyl4N+Br- (designated A+B- hereafter) in formamide are analyzed mass spectrometrically (MS) following mobility selection in ambient air in a differential mobility analyzer (DMA). Most of the sharp mobility peaks seen are identified as (AB)(n)A+ clusters, with 0 < or = n < ot = 5. One anomalously abundant and mobile ion is identified as NH4+(AB)4. Six ions in the (AB)n(A+)2 series are also identified, completing and correcting earlier mobility data for singly and doubly charged ions up to masses of almost 9000 Da. The more mobile of two broad humps seen in the mobility spectrum includes m/z values approximately from 2500 up to 12,000 Da. It is formed primarily by multiply charged (AB)n(A+)z clusters with multiple ammonium bromide adducts. Because of overlapping of many peaks of different m/z and charge state z, only a few individual species can be identified by MS alone in this highly congested region. However, the spectral simplification brought about by mobility selection upstream of the MS reveals a series of broad modulations in m/z space, with all ions resolved in the second, third, ...sixth modulation being in charge states z = 2, 3, ...6, respectively. Extrapolation of this trend beyond the sixth wave fixes the ion charge state (in some cases up to z = 15) and mass (beyond m = 175,000 u). This wavy structure had been previously observed and explained in terms of ion evaporation kinetics from volatile drops, though without mass identification. All observations indicate that the clusters are formed as charged residues, but their charge state is fixed by the Iribarne-Thomson ion evaporation mechanism. Consequently, the measured curve of cluster diameter versus z yields the two parameters governing ion evaporation kinetics. Clusters with z > 1 and electrical mobility Z > 0.495 cm2/V/s are metastable and evaporate a singly charged cluster, probably (AB)2A+, between the DMA and the MS. Plotting the electrical mobilities Z of the clusters in the form (z/Z)1/2 versus m(1/3) (both proportional to cluster diameter) collapse the data for all cluster sizes and charge states into one single straight line for Z below 0.495 cm2/V/s. This linear relation reveals a uniform apparent cluster density of 0.935 g/cm3 and an effective hard-sphere diameter of the air molecules of 0.44 nm. An anomalous mobility increase is observed at diameters below 3 nm.     

Behaviour of tetraalkylammonium ions in high‐field asymmetric waveform ion mobility spectrometry

High‐field asymmetric waveform ion mobility spectrometry (FAIMS) is an ion‐filtering technique recently adapted for use with liquid chromatography/mass spectrometry (LC/MS) to remove interferences during analysis of complex matrices. This is the first systematic study of a series of singly charged tetraalkylammonium ions by FAIMS‐MS. The compensation voltage (CV) is the DC offset of the waveform which permits the ion to emerge from FAIMS and it was determined for each member of the series under various conditions. The electrospray ionization conditions explored included spray voltage, vaporizer temperature, and sheath and auxiliary gas pressure. The FAIMS conditions explored included carrier gas flow rate, electrode temperature and composition of the carrier gas. Optimum desolvation was achieved using sufficient carrier gas (flow rate ≥2 L/min) to ensure stable response. Low‐mass ions (m/z 100–200) are more susceptible to changes in electrode temperature and gas composition than high mass ions (m/z 200–700). As a result of this study, ions are reliably analyzed using standard FAIMS conditions (dispersion voltage ?5000 V, carrier gas flow rate 3 L/min, 50% helium/50%nitrogen, inner electrode temperature 70°C and outer electrode temperature 90°C). Variation of FAIMS conditions may be of great use for the separation of very low mass tetraalkylammonium (TAA) ions from other TAA ions. The FAIMS conditions do not appear to have a major effect on higher mass ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Correlation between the shape of the ion mobility signals and the stepwise folding process of polylactide ions

In the field of polymer characterization, the use of ion mobility mass spectrometry (IMMS) remains mainly devoted to the temporal separation of cationized oligomers according to their charge states, molecular masses and macromolecular architectures in order to probe the presence of different structures. When analyzing multiply charged polymer ions by IMMS, the most striking feature is the observation of breaking points in the evolution of the average collision cross sections with the number of monomer units. Those breaking points are associated to the folding of the polymer chain around the cationizing agents. Here, we scrutinize the shape of the arrival time distribution (ATD) of polylactide ions and associate the broadening as well as the loss of symmetry of the ATD signals to the coexistence of different populations of ions attributed to the transition from opened to folded stable structures. The observation of distinct distributions reveals the absence of folded/extended structure interconversion on the ion mobility time scale (1–10 ms) and then on the lifetime of ions within the mass spectrometer at room temperature. In order to obtain information on the possible interconversion between the different observed populations upon ion activation, we performed IM–IM–MS experiments (tandem ion mobility measurements). To do so, mobility‐selected ions were activated by collisions before a second mobility measurement. Interestingly, the conversion by collisional activation from a globular structure into a (partially) extended structure, i.e. the gas phase unfolding of the ions, was not observed in the energetic regime available with the used experimental setup. The absence of folded/extended interconversion, even upon collisional activation, points to the fact that the polylactide ions are ‘frozen’ in their specific 3D structure during the desolvation/ionization electrospray processes. Copyright © 2017 John Wiley & Sons, Ltd.     

Ion-induced nucleation of dibutyl phthalate vapors on spherical and nonspherical singly and multiply charged polyethylene glycol ions

Dibutyl phthalate vapor nucleation induced by positive polyethylene glycol (PEG) ions with controlled sizes and charges was experimentally studied. The ions were produced by electrospray ionization, classified in a high-resolution differential mobility analyzer, and studied in a nano condensation nucleus counter of the mixing type. Ionic radii of PEG varied from 0.52 to 1.56 nm, including from singly to quadruply charged ions. Some of these ions are fully stretched chains, other are spherical, and others have intermediate forms, all of them having been previously characterized by mobility and mass spectrometry studies. Activation of PEG1080(+2) requires a supersaturation almost as high as that required for small singly charged ions and higher than for PEG1080(+). This anomaly is explained by the Coulombic stretching of the ion into a long chain, where the two charged centers appear to be relatively decoupled from each other. The critical supersaturation for singly charged spherical ions falls below Thomson's (capillary) theory and even below the already low values seen previously for tetraheptyl ammonium bromide clusters. Spherical PEG4120(+2) falls close to the Thomson curve. The trends observed for slightly nonspherical PEG4120(+3) and highly nonspherical (but not quite linear) PEG4120(+4) are intermediate between those of multiply charged spheres and small singly charged ions.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A2 and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase A2 and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained. In addition to the dominant protein [M + H]+ ions, multimeric and multiply charged ions were also observed in the mass spectra. The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected. Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes. Mass accuracies of 0.1 and 0.3% were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants.     

Analysis of carrageenans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. I. kappa-Carrageenans.

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectra of small kappa-carrageenans are reported and discussed. MALDI spectra can be obtained in both positive and negative ion mode. In the absence of extraneous metal ions, positive ions are formed by the attachment of one Na(+) ion to the carrageenan, whereas for negative ions one Na(+) ion is detached from the sulfate group. Multiply charged species are not observed in MALDI. Intense ESI spectra can be obtained in negative ion mode and now multiply charged species are seen. Alkali exchange experiments show that in these small carrageenan anions one, but only one, alkali metal ion is bound in a bidentate coordination with two ionic sulfate groups. G2-type ab initio calculations on model ions HO(-) [M(+)] (-)OH (M = Li, Na, K, Cs), as well as arguments based on a simple Coulombic interaction model, show that the bidentate stabilization energy drops rapidly as the size of the alkali cation increases. Exchange of Na(+) with Li(+) leads to expulsion of the Na(+) ion generating, in ESI, intense multiply charged anions. An attempt is made to rationalize this behavior in terms of hydration effects.     

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.     

The analysis of industrial synthetic polymers by electrospray droplet impact/secondary ion mass spectrometry

Electrospray droplet impact (EDI)/secondary ion mass spectrometry (SIMS) is a new desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from the atmospheric‐pressure electrospray are accelerated in vacuum by several kV and impact the sample deposited on the metal substrate. In this study, several industrial synthetic polymers, e.g. polystyrene (PS) and polyethylene glycol (PEG) were analyzed by EDI/SIMS mass spectrometry. For higher molecular weight analytes, e.g. PS4000 and PEG4600, EDI/SIMS mass spectra could be obtained when cationization salts are added. For the polymers of lower molecular weights, e.g. PEG300 and PEG600, they could be readily detected as protonated ions without the addition of cationization agents. Anionized PS was also observed in the negative ion mode of operation when acetic acid was added to the charged droplet. Compared to matrix‐assisted laser desorption/ionization (MALDI), ion signal distribution with lower background signals could be obtained particularly for the low‐molecular weight polymers. Copyright © 2009 John Wiley & Sons, Ltd.