Mass and charge assignment for electrospray ions by cation adduction

The assignment of the mass (m) value from the m/z value for ions with a multiple number of charges (z) in electrospray mass spectra usually utilizes multiple peaks of the same m but different z values, or unit-mass—separated isotopic peaks of the same z value from high resolution spectra. The latter approach is also feasible with much less resolving power using adduct ions of much higher mass separation. The application of this to mixture spectra containing many masses, such as spectra from tandem mass spectrometry (MS/MS) ion dissociation, does not appear to have been pointed out previously. Thus, replacing two protons by one Cu²⁺ ion increases the mass by 61.5 Da, with this shift providing a mass scale for assignment of m and z from this pair of m/z values. The more common Na⁺ adduct peaks provide a 22.0 Da separation, of utility for 1000 resolving power only below approximately 10 kDa. Further, collisional dissociation lowers the degree of Cu²⁺ adduction in the resulting sequence-specific fragment ions much less than that of the corresponding Na⁺ adducts, making the Cu²⁺ adducts far more useful for m and z determination in MS/MS studies.     

Automated data reduction for hydrogen/deuterium exchange experiments,enabled by high-resolution fourier transform ion cyclotron resonance mass spectrometry

Mass analysis of proteolytic fragment peptides following hydrogen/deuterium exchange offers a general measure of solvent accessibility/hydrogen bonding (and thus conformation) of solution-phase proteins and their complexes. The primary problem in such mass analyses is reliable and rapid assignment of mass spectral peaks to the correct charge state and degree of deuteration of each fragment peptide, in the presence of substantial overlap between isotopic distributions of target peptides, autolysis products, and other interferant species. Here, we show that at sufficiently high mass resolving power (m/Δm_50% ≥ 100,000), it becomes possible to resolve enough of those overlaps so that automated data reduction becomes possible, based on the actual elemental composition of each peptide without the need to deconvolve isotopic distributions. We demonstrate automated, rapid, reliable assignment of peptide masses from H/D exchange experiments, based on electrospray ionization FT-ICR mass spectra from H/D exchange of solution-phase myoglobin. Combined with previously demonstrated automated data acquisition for such experiments, the present data reduction algorithm enhances automation (and thus expands generality and applicability) for high-resolution mass spectrometry-based analysis of H/D exchange of solution-phase proteins.     

Polydimethylsiloxane-based wide-range mass calibration for direct analysis in real-time mass spectrometry

Direct analysis in real-time mass spectrometry (DART-MS) is normally applied for small-molecule analysis up to about m/z 1,000. Here, for the analysis of polydimethylsiloxanes, high-mass capabilities expanding beyond m/z 3,000 are demonstrated. In addition, polydimethylsiloxanes provide an ideal mass calibration standard for positive-ion DART-MS. A mass reference list has been compiled to cover ions from m/z 200 up to m/z 2,600. Species with more than 20 silicon atoms exhibit increasingly broader isotopic patterns with decreasing abundances of the monoisotopic ions. The use of the first isotopic peaks for analyte ions above m/z 2,000 serves as a work-around and ensures easy and reproducible recognition of the reference peaks by the instrument data system. Here, the positive-ion DART mass spectra of polydimethylsiloxanes and the corresponding experimental procedures are described, and the mass reference list is provided.     

Charge reduction lowers mass resolving power for isotopically resolved electrospray ionization Fourier transform ion cyclotron resonance mass spectra

Electrospray ionization of synthetic or biological macromolecules above ∼1–2 kDa in mass typically produces ions of multiple charge states. Several recent papers have illustrated charge reduction as a means to simplify low-resolution electrospray ionization mass spectra, at the cost of significant loss in signal-to-noise ratio. However, if mass resolving power is sufficiently high (as in Fourier transform ion cyclotron resonance mass spectrometry) to resolve the heavy-atom isotopic distribution, then charge reduction actually lowers mass resolving power by a factor proportional to the ion charge. For proteins or nucleic acids of 10–50 kDa in mass, reducing the charge state to unity thus lowers mass resolving power by a factor of 10–50. In other words, as long as it is possible to resolve the isotopic distributions, charge reduction has no advantages for electrospray ionization mass spectrometry and has the very serious disadvantage of greatly degraded mass resolving power. Copyright © 2001 John Wiley & Sons, Ltd.     

Advantages of high-resolution and high-mass range magnetic-sector mass spectrometry for electrospray ionization

Electrospray ionization (ESI) mass spectra have been measured on a magnetic-sector double-focusing mass spectrometer for a number of proteins and peptides. It is pointed out how in theory raising the mass resolution of a mass spectrometer from 800–1000 to 2400–3000 significantly increases the precision with which the envelope of isotopic peaks of a protein ion (or other organic ion) can be defined, particularly at higher masses. Better definition of the isotopic envelope ought to lead to higher precision in the experimental determination of molecular mass, which has been demonstrated. It is shown how ESI mass spectra of high-mass molecules are significantly less congested at higher m/z values, so that for these molecules (RMM > 40 000) there is an advantage in being able to record peaks at higher m/z values (m/z > 2000) representing ions with fewer charges. Fragmentation of a small peptide in the ESI source has been found to provide sequence information.     

Instrument and process independent binning and baseline correction methods for liquid chromatography–high resolution-mass spectrometry deconvolution

Setting appropriate bin sizes to aggregate hyphenated high-resolution mass spectrometry data, belonging to similar mass over charge (m/z) channels, is vital to metabolite quantification and further identification. In a high-resolution mass spectrometer when mass accuracy (ppm) varies as a function of molecular mass, which usually is the case while reading m/z from low to high values, it becomes a challenge to determine suitable bin sizes satisfying all m/z ranges. Similarly, the chromatographic process within a hyphenated system, like any other controlled processes, introduces some process driven systematic behavior that ultimately distorts the mass chromatogram signal. This is especially seen in liquid chromatogram–mass spectrometry (LC–MS) measurements where the gradient of the solvent and the washing step cycle—part of the chromatographic process, produce a mass chromatogram with a non-uniform baseline along the retention time axis. Hence prior to any automatic signal decomposition techniques like deconvolution, it is a equally vital to perform the baseline correction step for absolute metabolite quantification. This paper will discuss an instrument and process independent solution to the binning and the baseline correction problem discussed above, seen together, as an effective pre-processing step toward liquid chromatography–high resolution-mass spectrometry (LC–HR-MS) data deconvolution.     

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10^?9 torr. The increased pumping speed attainable with cryopumping (> 10⁵ L/s) allowed brief pressure excursions to above 10^?4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M_r 5734) and ubiquitin (M_r 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H₃PO₄ adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4⁺ charge state (m/z 1434) of insulin.     

Elucidating collision induced dissociation products and reaction mechanisms of protonated uracil by coupling chemical dynamics simulations with tandem mass spectrometry experiments

In this study we have coupled mixed quantum‐classical (quantum mechanics/molecular mechanics) direct chemical dynamics simulations with electrospray ionization/tandem mass spectrometry experiments in order to achieve a deeper understanding of the fragmentation mechanisms occurring during the collision induced dissociation of gaseous protonated uracil. Using this approach, we were able to successfully characterize the fragmentation pathways corresponding to ammonia loss (m/z 96), water loss (m/z 95) and cyanic or isocyanic acid loss (m/z 70). Furthermore, we also performed experiments with isotopic labeling completing the fragmentation picture. Remarkably, fragmentation mechanisms obtained from chemical dynamics simulations are consistent with those deduced from isotopic labeling. Copyright © 2015 John Wiley & Sons, Ltd.     

Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry

Electrospray ionization with a forward-geometry magnetic sector mass spectrometer was used for collisionally activated dissociation studies of multiply charged polypeptides and for studying non-covalently bound protein systems. The high-resolution capabilities of a high-performance instrument allow the resolution of isotopic contributions for product ions and molecular ion species. Determination of product ion charge states by this method reduces difficulties in the interpretation of product ion mass spectra from multiply charged precursors, which are generated either in the atmospheric pressure/vacuum electrospray interface or in the collision chamber of the mass spectrometer. Extended tandem mass spectrometric experiments have the potential for sequencing larger polypeptides. However, evidence for isomerization of gas-phase product ions from substance P and substance P analogues was observed, complicating the interpretation of product ion spectra. Non-covalent complexes can also be studied by electrospray ionization magnetic sector MS. The higher m/z range of such an instrument is a major advantage for studying weakly bound systems, such as heme–protein systems (myoglobin, hemoglobin) and protein aggregates (concanavalin A), because of their tendency to form complex ions with relatively low charge states.     

A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra

This article describes a new algorithm for charge state determination and deconvolution of electrospray ionization (ESI) mass-to-charge ratio spectra. The algorithm (Zscore) is based on a charge scoring scheme that incorporates all above-threshold members of a family of charge states or isotopic components, and deconvolves both low- and high-resolution mass-to-charge ratio spectra, with or without a peak list (stick plot). A scoring weight factor, log (I/I ₀), in which I is the signal magnitude at a calculated mass-to-charge ratio, and I ₀ is the signal threshold near that mass-to-charge ratio, was used in most cases. For high-resolution mass-to-charge ratio spectra in which all isotopic peaks are resolved, the algorithm can deconvolve overlapped isotopic multiplets of the same or different charge state. Compared to other deconvolution techniques, the algorithm is robust, rapid, and fully automated (i. e., no user input during the deconvolution process). It eliminates artifact peaks without introducing peak distortions. Its performance is demonstrated for experimental ESI Fourier transform ion cyclotron resonance mass-to-charge ratio spectra (both low and high resolution). Charge state deconvolution to yield a “zero-charge” mass spectrum should prove particularly useful for interpreting spectra of complex mixtures, identifying contaminants, noncovalent adducts, fragments (N-terminal, C-terminal, internal), and chemical modifications of electrosprayed biomacromolecules.     

Automated charge state determination of complex isotope-resolved mass spectra by peak-target Fourier transform

This study describes a new algorithm for charge state determination of complex isotope-resolved mass spectra. This algorithm is based on peak-target Fourier transform (PTFT) of isotope packets. It is modified from the widely used Fourier transform method because Fourier transform may give ambiguous charge state assignment for low signal-to-noise ratio (S/N) or overlapping isotopic clusters. The PTFT algorithm applies a novel "folding" strategy to enhance peaks that are symmetrically spaced about the targeted peak before applying the FT. The "folding" strategy multiplies each point to the high-m/z side of the targeted peak by its counterpart on the low-m/z side. A Fourier transform of this "folded" spectrum is thus simplified, emphasizing the charge state of the "chosen" ion, whereas ions of other charge states contribute less to the transformed data. An intensity-dependent technique is also proposed for charge state determination from frequency signals. The performance of PTFT is demonstrated using experimental electrospray ionization Fourier transform ion cyclotron resonance mass spectra. The results show that PTFT is robust for charge state determination of low S/N and overlapping isotopic clusters, and also useful for manual verification of potential hidden isotopic clusters that may be missed by the current analysis algorithms, i.e., AID-MS or THRASH.     

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Calibration of mass ranges up to m/z 10,000 in electrospray mass spectrometers

We report that the cesium salts of monobutyl phthalate, heptafluorobutyric acid, tridecafluoroheptanoic acid, and perfluorosebacic acid generate salt/cluster ions that provide calibration peaks for electrospray mass spectrometers. In the case of the cesium salt of tridecafluoroheptanoic acid ions are available up to m/z 10,000.     

Optimised Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI-MSI) for the Analysis of Proteins/Peptides Directly from Tissue Sections on a Travelling Wave Ion Mobility Q-ToF

Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) is typically known for the ionisation of small molecules such as lipids and metabolites, in singly charged form. Here we present a method that allows the direct detection of proteins and peptides in multiply charged forms directly from tissue sections by DESI. Utilising a heated mass spectrometer inlet capillary, combined with ion mobility separation (IMS), the conditions with regard to solvent composition, nebulising gas flow, and solvent flow rate have been explored and optimised. Without the use of ion mobility separation prior to mass spectrometry analysis, only the most abundant charge series were observed. In addition to the dominant haemoglobin subunit(s) related trend line in the m/z vs drift time (DT) 2D plot, trend lines were found relating to background solvent peaks, residual lipids and, more importantly, small proteins/large peptides of lower abundance. These small proteins/peptides were observed with charge states from 1+ to 12+, the majority of which could only be resolved from the background when using IMS. By extracting charge series from the 2D m/z vs DT plot, a number of proteins could be tentatively assigned by accurate mass. Tissue images were acquired with a pixel size of 150 μm showing a marked improvement in protein image resolution compared to other liquid-based ambient imaging techniques such as liquid extraction surface analysis (LESA) and continuous-flow liquid microjunction surface sampling probe (LMJ-SSP) imaging.

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Selective detection of unknown organic bromine compounds and quantification potentiality by negative-ion electrospray ionization mass spectrometry with induced in-source fragmentation

For the detection of unknown organic bromine compounds, a liquid chromatography–mass spectrometry (LC-MS) method with negative-ion electrospray ionization (NI-ESI) and induced in-source fragmentation (IISF) was established. After LC separation, the molecules are fragmentized in the source, and bromide is detected via m/z 79 and m/z 81 based on the isotopic occurrence of bromine. In this way, the retention times of the unknown organobromine compounds are determined, and this can be used to extract additional structural information (number of bound bromine atoms, molecular mass and fragmentation scheme) from measurements in the commonly used but less sensitive scan mode. The analysis of known organobromine compounds shows that LC/NI-ESI-IISF mass spectrometry with detection of m/z 79 and 81 is more sensitive than the detection of daughter ions (LC/ESI/MS-MS). Therefore, we present a method not only for the detection of unknown organic bromine compounds, but also for the selective and sensitive detection and quantification of known organobromine compounds.     

Performance evaluation of a high-field orbitrap mass analyzer

A new design of the Orbitrap^™ mass analyzer is presented. Higher frequencies of ion oscillations and hence higher resolving power over fixed acquisition time are achieved by decreasing the gap between the inner and outer Orbitrap electrodes, thus providing higher field strength for a given voltage. Experimental results confirm maximum FWHM resolving power in excess of 350,000 at m/z 524 and 600,000 at m/z 195, isotopic resolution of proteins above 40 kDa, and a single-shot dynamic range of 25,000. It was also found that mass shifts in the new design depend very little on space charge inside the analyzer. This performance was achieved using higher voltages and by careful balancing of construction tolerances and operation parameters, which appeared to vary in narrower ranges of tuning than for a standard Orbitrap analyzer.     

Screening DNA Adducts by LC–ESI–MS–MS: Application to Screening New Adducts Formed from Acrylamide

A method for screening DNA adducts with unknown chemical structures was developed; it involves the use of liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI–MS–MS). In electrospray ionization (ESI) product ion mass spectra of guanine adducts, fragment ions were observed at m/z 152 and 135. Precursor ion scan analysis of these fragment ions indicated that the screening of DNA adducts would be possible. The developed method was used for the analysis of DNA adducts derived from acrylamide, which is not only a constituent of many commonly consumed foods but also a carcinogenic compound. We successfully discovered new guanine adducts. The results of this study indicate that the developed method is useful for screening new DNA adducts.     

Twelve Million Resolving Power on 4.7 T Fourier Transform Ion Cyclotron Resonance Instrument with Dynamically Harmonized Cell—Observation of Fine Structure in Peptide Mass Spectra

Resolving power of about 12,000 000 at m/z 675 has been achieved on low field homogeneity 4.7 T magnet using a dynamically harmonized Fourier transform ion cyclotron resonance (FT ICR) cell. Mass spectra of the fine structure of the isotopic distribution of a peptide were obtained and strong discrimination of small intensity peaks was observed in case of resonance excitation of the ions of the whole isotopic cluster to the same cyclotron radius. The absence of some peaks from the mass spectra of the fine structure was explained basing on results of computer simulations showing strong ion cloud interactions, which cause the coalescence of peaks with m/z close to that of the highest magnitude peak. The way to prevent peak discrimination is to excite ion clouds of different m/z to different cyclotron radii, which was demonstrated and investigated both experimentally and by computer simulations.

Boundaries of Mass Resolution in Native Mass Spectrometry

Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.

Desorption electrospray ionization–mass spectrometric analysis of low vapor pressure chemical particulates collected from a surface

The collection of a low vapor pressure chemical simulant triethyl phosphate sorbed onto silica gel (TEP/SG) from a surface with subsequent analysis of the TEP/SG particulates using desorption electrospray ionization–mass spectrometry (DESI–MS) is described. Collection of TEP/SG particulates on a surface was accomplished using a sticky screen sampler composed of a stainless steel screen coated with partially polymerized polydimethylsiloxane (PDMS). DESI–MS analysis of TEP/SG particulates containing different percentages of TEP sorbed onto silica gel enabled the generation of response curves for the TEP ions m/z 155 and m/z 127. Using the response curves the calculation of the mass of TEP in a 25 wt% sample of TEP/SG was calculated, results show that the calculated mass of TEP was 14% different from the actual mass of TEP in the sample using the m/z 127 TEP ion response curve. Detection limits for the TEP vapor and TEP/SG particulates were calculated to be 4 μg and 6 particles, respectively.