Improved mass accuracy for tandem mass spectrometry

With the emergence of top-down proteomics, the ability to achieve high mass measurement accuracy on tandem MS/MS data will be beneficial for protein identification and characterization. (FT-ICR) Fourier transform ion cyclotron resonance mass spectrometers are the ideal instruments to perform these experiments with their ability to provide high resolution and mass accuracy. A major limitation to mass measurement accuracy in FT-ICR instruments arises from the occurrence of space charge effects. These space charge effects shift the cyclotron frequency of the ions, which compromises the mass measurement accuracy. While several methods have been developed that correct these space charge effects, they have limitations when applied to MS/MS experiments. It has already been shown that additional information inherent in electrospray spectra can be used for improved mass measurement accuracy with the use of a computer algorithm called DeCAL (deconvolution of Coulombic affected linearity). This paper highlights a new application of the strategy for improved mass accuracy in tandem mass analysis. The results show a significant improvement in mass measurement accuracy on complex electron capture dissociation spectra of proteins. We also demonstrate how the improvement in mass accuracy can increase the confidence in protein identification from the fragment masses of proteins acquired in MS/MS experiments.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Charge remote fragmentation of fatty acids cationized with alkaline earth metal ions

Fatty acids can be collisionally activated as [M ? H + Cat]⁺, where Cat is an alkaline earth metal, by using tandem mass spectrometry. High-energy collisional activation induces charge remote fragmentation to give structural information. In the full scan mass spectra molecular ions are easily identified, particularly when barium is used as a cationizing agent; ions are shifted to a higher mass, lower chemical noise region of the mass spectrum. Moreover, the isotopic pattern of barium is characteristic, and the high mass defect of barium allows an easy separation of the cationized analyte from any remaining interfering ions (chemical noise), provided medium mass-resolving power is available. An additional advantage is that most of the ion current is localized in [M ? H + Cat]⁺ species. Structural analysis of fatty acids can be performed when the sample size is as low as 1 ng.     

The modern mass spectrometer—a complete chemical laboratory

Using nitrobenzene as an example, various ways in which a contemporary mass spectrometer can be utilized to yield a wealth of information about the compound studied are reviewed. Applying a variety of different techniques and procedures, in addition to the conventional low resolution mass spectrum, the following nitrobenzene spectra have been obtained: collision induced dissociation mass spectrum, mass analysed ion kinetic energy spectra, collision induced dissociation mass analysed ion kinetic energy spectra, spectra obtained at constant B/E, spectra obtained at constant B²/E, high voltage scans of metastable ion fragmentation processes, consecutive fragmentations in different field free regions, charge exchange mass spectra, charge stripping mass spectra, doubly charged ion mass spectra, chemical ionization mass spectra, negative ion mass spectra, negative ion mass analysed ion kinetic energy spectra, negative ion mass analysed ion kinetic energy collision induced dissociation spectra, charge inversion spectra, etc. The complementary types of information available from the above studies are discussed to show the unique versatility of mass spectrometry as a technique for the examination of organic compounds.     

Weighing synthetic polymers of ultra‐high molar mass and polymeric nanomaterials: What can we learn from charge detection mass spectrometry?

Advances in soft ionization techniques for mass spectrometry (MS) of polymeric materials make it possible to determine the masses of intact molecular ions exceeding megadaltons. Interfacing MS with separation and fragmentation methods has additionally led to impressive advances in the ability to structurally characterize polymers. Even if the gap to the megadalton range has been bridged by MS for polymers standards, the MS‐based analysis for more complex polymeric materials is still challenging. Charge detection mass spectrometry (CDMS) is a single‐molecule method where the mass and the charge of each ion are directly determined from individual measurements. The entire molecular mass distribution of a polymer sample can be thus accurately measured. Described in this perspective paper is how molecular weight distribution as well as charge distribution can provide new insights into the structural and compositional studies of synthetic polymers and polymeric nanomaterials in the megadalton to gigadalton range of molecular weight. The recent multidimensional CDMS studies involving couplings with separation and dissociation techniques will be presented. And, finally, an outlook for the future avenues of the CDMS technique in the field of synthetic polymers of ultra‐high molar mass and polymeric nanomaterials will be provided.     

Characterization of heteroscedastic measurement noise in the absence of replicates

A method is described for the characterization of measurement errors with non-uniform variance (heteroscedastic noise) in contiguous signal vectors (e.g., spectra, chromatograms) that does not require the use of replicated measurements. High-pass digital filters based on inverted Blackman windowed sinc smoothing coefficients are employed to provide point estimates of noise from measurement vectors. Filter parameters (number of points, cutoff frequency) are selected based on the amplitude spectrum of the signal in the Fourier domain. Following this, noise estimates from multiple signals are partitioned into bins based on a variable that correlates with the noise amplitude, such as measurement channel or signal intensity. The noise estimates in each bin are combined to estimate the standard deviation and, where appropriate, a functional model of the noise can be obtained to characterize instrumental errors (e.g., shot noise, proportional noise). The proposed method is demonstrated and evaluated with both simulated and experimental data sets, and results are compared with replicated measurements. Experimental data includes fluorescence spectra, ion chromatograms from liquid chromatography/mass spectrometry, and UV–vis absorbance spectra. The limitations and advantages of the new method compared to replicate analysis are presented.     

Applications of charge stripping/charge exchange spectra of organic ions

The application of a new technique which involves the combination of charge stripping and charge exchange processes has been investigated. Ions are charge-stripped in the second field-free region of a triple-sector mass spectrometer (BEE geometry), and then subjected to a charge exchange reaction in the third field-free region. The resulting charge stripping/charge exchange (CS/CE) spectrum is free from interference, which is otherwise common in charge stripping spectra. Comparisons between charge stripping and CS/CE spectra are made in cases where both kinds of spectra are obtainable. In order to assess the applicability of this new technique to studies of isomeric ion structures, species for which charge stripping spectra have previously been unobtainable have been chosen. CS/CE spectra of [C₆H₆]⁺˙, [C₆H₅]⁺, [C₈H₁₀]⁺˙ and [C₇H₇O]⁺ ions from a variety of precursors are recorded: in most cases, sufficient differences are observed to permit distinction between isomeric structures (or mixtures of structures). Previous studies which have shown that stable doubly charged molecular ions of ethane cannot be formed by the charge stripping technique are confirmed from its CS/CE spectrum.     

Interpretation of electrospray/ion trap mass spectra of bile acids and other surfactants

Electrospray spectra of various bile acids and other surfactants were obtained using an ion trap instrument. Bile acids and bile acid derivatives such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) self associate to form micelles in a stepwise process. Their spectra show a distinct pattern of aggregation, with ions evenly separated in the m/z space. A new parameter, n/z, can be used to characterize such ions, where n is the number of molecules and z is the charge of the aggregate. The values of z were determined using multi-stage mass spectrometry (MS(n)) and high resolution in the ion trap.     

Spectral transformations for deconvolution methods applied on gas chromatography-mass spectrometry data

In hyphenated chromatography, overlapping chromatographic peaks can be resolved into pure spectra and pure chromatographic profiles by several multivariate deconvolution techniques. In general, these methods require bilinearity, which implies that the spectrum of each analyte is constant. The slow scan speeds normally used in gas chromatography-mass spectrometry (GC-MS) will destroy bilinearity and introduce systematic noise in the data because the concentration in the detector changes during the scan. This effect, described as the scan effect, may hinder successful resolution by multivariate deconvolution. In selected ion monitoring (SIM) GC-MS, the scan effect may be removed by simple transformations of the mass spectra. The effects of different transformations are demonstrated both on pure chromatographic peaks and on difficult resolution problems where there are small differences between the spectra of the analytes.     

Determination of peptide and protein ion charge states by fourier transformation of isotope-resolved mass spectra

We report an automated method for determining charge states from high-resolution mass spectra. Fourier transforms of isotope packets from high-resolution mass spectra are compared to Fourier transforms of modeled isotopic peak packets for a range of charge states. The charge state for the experimental ion packet is determined by the model isotope packet that yields the best match in the comparison of the Fourier transforms. This strategy is demonstrated for determining peptide ion charge states from "zoom scan" data from a linear quadrupole ion trap mass spectrometer, enabling the subsequent automated identification of singly- through quadruply-charged peptide ions, while reducing the numbers of conflicting identifications from ambiguous charge state assignments. We also apply this technique to determine the charges of intact protein ions from LC-FTICR data, demonstrating that it is more sensitive under these experimental conditions than two existing algorithms. The strategy outlined in this paper should be generally applicable to mass spectra obtained from any instrument capable of isotopic resolution.     

Space charge effects on relative peak heights in fourier transform-ion cyclotron resonance spectra

Ion trajectory calculations have confirmed that space charge interactions can be a source for mass discrimination seen in Fourier transform-ion cyclotron resonance (FT-ICR) spectra. As compared with the previously recognized mechanism of z-axis excitation, ion-ion repulsion is a mechanism which specifically affects relative peak heights of ions close in mass, and is most severe for low excitation radiofrequency (rf) amplitudes. In this mechanism, Coulomb repulsion significantly perturbs the motion of the ion clouds during excitation and alters the final cyclotron orbital radii. Under these conditions peak heights do not accurately reflect the true ion abundances in the FT-ICR spectrometer. Mass discrimination can be minimized by using low numbers of ions, low ion densities, and a short, high amplitude rf excitation waveform. Experimental observation of the relative peak heights of the m/z 91, 92, and 134 ions in n-butylbenzene gives quantitative confirmation of the results of the trajectory calculations. Chirp, SWIFT, and impulse excitation were modeled: impulse excitation was found to be most effective in minimizing the effects of space charge interactions.     

A new maximum likelihood approach with asymmetric residual distribution for multicomponent mass spectra analysis

This paper proposes a new maximum likelihood approach for the deconvolution of identity and quantity of individual compounds based on the multicomponent mass spectra measured by mass spectrometry (MS). Mixture analysis of multicomponent mass spectra is, typically, based on a linear multicomponent mass spectrum model, where the compounds of the measured spectra to be solved are explicitly stated and assumed to be known. In many cases, however, the measured spectrum may contain unknown compounds that are not explicitly stated in the model and a commonly used least square (LS) solution fails. Moreover, a standard improvement over the LS method in these cases, namely the M-estimation (ME) approach, also suffers from this same problem. Our method overcomes the limitations of the LS and ME methods by modeling the effect of the unknown compound(s) to the residual of the linear model. The experimental results presented show that this new approach can separate more robustly the complex multicomponent mass spectra into their individual constituents compared to the LS and ME methods.     

Nanoelectrospray—More than just a minimized-flow electrospray ionization source

The comparison between electrospray ionization (ESI) mass spectra from NaCl solutions with and without analyte obtained under ionspray and nanospray conditions reveals different mass spectral behavior of the two ESI techniques. This can be attributed to the different initial droplet sizes which are in the microns range for ionspray, while in nanospray they are believed to be about one order of magnitude smaller. In the context of the widely accepted uneven-fission model, nanospray would then enter one fission generation later; in addition, a higher initial droplet surface charge density in nanospray results in early fissions without extensive evaporation and thus increase in sample and salt concentration. This rationalizes that ionspray spectra closely resemble nanospray spectra from solutions with about one order of magnitude higher salt concentrations, showing a higher tolerance of nanospray towards salt contamination. When the analyte is a peptide (in a solution containing a high molar surplus of salt), molecule ion formation effectively competes with salt cluster ion formation; when the analyte is a sugar, it is detectable beside a high salt concentration only with nanospray, indicating the supporting effect of surface activity on ion release in the case of peptides. A model is presented which explains the different mass spectral behaviour of ionspray and nanospray by suggesting different "predominant fission pathways" depending on the size of the initial droplets.     

A fast method for the measurement of diffusion coefficients: one‐dimensional DOSY

A new experiment for the single‐scan measurement of diffusion coefficients is presented. The principle is to introduce a spatial variation in the parameters of a conventional pulse sequence, so that all of the scans required to determine some physical parameter can be recorded simultaneously from different parts of the sample. The spectrum is acquired in the presence of a weak read gradient so that the resulting lineshapes contain the information required. The pulse sequence is described in detail and demonstrated on a sample containing three components; its advantages and limitations are discussed in relation to those of existing techniques. For uncrowded spectra with high signal‐to‐noise ratio, this experiment provides an order of magnitude reduction in experiment time compared with conventional methods and is likely to be of most benefit where samples are changing rapidly with time or where a long period of polarization, which may be difficult to reproduce accurately, prohibits the use of multiple‐scan techniques. Copyright © 2003 John Wiley & Sons, Ltd.     

The analysis of polystyrene and polystyrene aggregates into the mega dalton mass range by cryodetection MALDI TOF MS

Mass spectra of atactic polystyrene were collected into the mega-dalton mass range with a matrix-assisted laser desorption ionization time of flight (MALDI TOF) mass spectrometer, which incorporates a cryodetector comprised of an array of 16 superconducting tunnel junctions (STJ). The STJ cryodetector, theoretically, has no loss in signal response at any mass compared with the reduced signal found at high mass when using a conventional secondary-ionization detector. Since ion detection at high m/z is one of the fundamental limitations of mass spectrometry (MS), the cryodetector was used to explore the high m/z limit of the MALDI TOF technique for the analysis of two polymer types. Mass spectra were collected for polystyrene at Mn 170, 400, 900, and 2000 kDa and polymethyl methacrylate (PMMA) at Mn 62.6 kDa and 153.7 kDa. For polystyrene, the data showed a trend toward increased aggregation and charge state with mass. The Mn 2 MDa polystyrene data revealed a peak at m/z 2.2 MegaTh and a charge state analysis revealed that these ions were primarily polystyrene aggregates with a mass of approximately 4 MDa. This aggregate assignment was possible because the cryodetector response allows for the determination of a charge state up to about four. The contribution of each charge state for a selected peak can be determined in this fashion. This analysis revealed the preferential formation of doubly charged even-numbered aggregates over odd-numbered aggregates for high molecular mass polystyrene. A potential mechanism for the aggregation process for doubly charged species is discussed.     

Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument

The automated use of a matrix-assisted laser desorption ionization (MALDI) mass spectrometer (MS) is described for image analysis of samples through implementation of new software for instrument control, data acquisition, and data analysis. The software permits automated acquisition of MS MALDI spectra to form an ordered data array and contains display features to provide images at one or more mass-to-charge ratio values. The technique can be used to scan tissue samples, blotted samples, gels, or other sample surfaces where the image analysis of that sample is required. The program achieves a time of typically 1 s per image point, permitting an analysis made up of large numbers of points with high spatial resolution up to 850 dpi. The features of the software are demonstrated in this paper with samples of printed images, where visible images can be compared to those obtained by mass spectrometry. Quantitative aspects are introduced by analyzing a series of sample spots containing different amounts of several proteins.     

Mass and charge state assignment for proteins and peptide mixtures via noncovalent adduction in electrospray mass spectrometry

A method has been developed that takes advantage of the formation of noncovalent compounds in electrospray mass spectrometry. Mixtures of proteins and peptides are shown to produce an intense ion that corresponds to a 1:1 complex with a crown ether (18-crown-6). Although the crown ether may be added directly to the solution, for the current experiments it is introduced via the methanol liquid sheath. The spacing of these complexed species in the mass spectrum allows unambiguous determination of the charge state of the ions and their actual mass. Through constant neutral loss scans, charge state may be determined, mass assigned, spectra simplified, and chemical noise may be reduced for the analysis of complex peptide samples without chromatographic separation. Finally, the prevalence of single complexation permits mass assignments based on the mass difference of a single protein ion and its complexed form at any charge state. In essence, the method performs a separation based on charge state. It can be used to complement chromatographic separation and deconvolution algorithms for the electrospray mass spectrometry analysis of peptide-protein mixtures.     

Wavelet-Based Method for Time-Domain Noise Analysis and Reduction in a Frequency-Scan Ion Trap Mass Spectrometer

We adopt an orthogonal wavelet packet decomposition (OWPD) filtering approach to cancel harmonic interference noises arising from an AC power source in time domain and remove the resulting rf voltage interference noise from the mass spectra acquired by using a charge detection frequency-scan quadrupole ion trap mass spectrometer. With the use of a phase lock resampling technique, the transform coefficients of the rf interference in signals become a constant, exhibiting a shift of the baseline in different rf phases. The rf interference is therefore removable by shifting the baselines back to zero in OWPD coefficients. The approach successfully reduces the time-domain background noise from 1367 electrons (rms) to 408 electrons (rms) (an improvement of 70?%) and removes the high frequency noise components in the charge detection ion trap mass spectrometry. Unlike other smoothing or averaging methods commonly used in the mass-to-charge (m/Ze) domain, our approach does not cause any distortion of original signals.     

A Combined Desorption Ionization by Charge Exchange (DICE) and Desorption Electrospray Ionization (DESI) Source for Mass Spectrometry

A source that couples the desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) techniques together was demonstrated to broaden the range of compounds that can be analyzed in a single mass spectrometric experiment under ambient conditions. A tee union was used to mix the spray reagents into a partially immiscible blend before this mixture was passed through a conventional electrospray (ES) probe capillary. Using this technique, compounds that are ionized more efficiently by the DICE method and those that are ionized better with the DESI procedure could be analyzed simultaneously. For example, hydroquinone, which is not detected when subjected to DESI-MS in the positive-ion generation mode, or the sodium adduct of guaifenesin, which is not detected when examined by DICE-MS, could both be detected in one experiment when the two techniques were combined. The combined technique was able to generate the molecular ion, proton and metal adduct from the same compound. When coupled to a tandem mass spectrometer, the combined source enabled the generation of product ion spectra from the molecular ion and the [M + H]⁺ or [M + metal]⁺ ions of the same compound without the need to physically change the source from DICE to DESI. The ability to record CID spectra of both the molecular ion and adduct ions in a single mass spectrometric experiment adds a new dimension to the array of mass spectrometric methods available for structural studies.     

Improved fourier-transform ion-cyclotron-resonance mass spectrometry of large biomolecules

Initial results from a Fourier-transform mass spectrometer with a 6.2 Tesla magnet using electrospray ionization show substantial improvements in resolution, mass accuracy, mass range, signal/noise, and tandem mass spectromehy capabilities compared to our earlier 2.8 T instrument that demonstrated the first unit resolution mass spectra of molecules as large as myoglobin (17 kDa). The new instrument exhibits greater than 10⁶ and 10⁵ resolving power for 8.6 and 29 kDa, respectively, proteins. Using an internal standard, the mass measuring error for myoglobin is less than 1 ppm. Nozzle-skimmer dissociation during electrospray of carbonic anhydrase (29 kDa) has yielded 38 fragment ions for which both mass and charge are identifiable; of these, 21 have been assigned to expected oligopeptide fragments.