Reduction of chemical formulas from the isotopic peak distributions of high-resolution mass spectra

A method has been developed for the reduction of the chemical formulas of compounds in complex mixtures from the isotopic peak distributions of high-resolution mass spectra. The method is based on the principle that the observed isotopic peak distribution of a mixture of compounds is a linear combination of the isotopic peak distributions of the individual compounds in the mixture. All possible chemical formulas that meet specific criteria (e.g., type and number of atoms in structure, limits of unsaturation, etc.) are enumerated, and theoretical isotopic peak distributions are generated for each formula. The relative amount of each formula is obtained from the accurately measured isotopic peak distribution and the calculated isotopic peak distributions of all candidate formulas. The formulas of compounds in simple spectra, where peak components are fully resolved, are rapidly determined by direct comparison of the calculated and experimental isotopic peak distributions. The singular value decomposition linear algebra method is used to determine the contributions of compounds in complex spectra containing unresolved peak components. The principles of the approach and typical application examples are presented. The method is most useful for the characterization of complex spectra containing partially resolved peaks and structures with multiisotopic elements.     

Large Molecule Characterization Based upon Individual Ion Detection with Electrospray Ionization-FTICR Mass Spectrometry

We report a new method for mass spectrometric measurements of high-molecular-weight species based on the summation of sequential Fourier transform ion cyclotron resonance (FTICR) spectra of individual multiply charged ions. This approach produces statistically useful mass spectra for large multiply charged molecular species formed by electrospray ionization and circumvents conventional limitations upon achievable resolving power and precision for high-molecular-weight species which arise due to Coulombic constraints. For very large molecules with tens to thousands of charges each, the total number of charges required to define the charge-state distribution, and thus provide accurate mass information, greatly exceeds the useful charge capacity of the FTICR cell. As trapped ion populations approach or exceed this capacity, FTICR performance degrades due to large frequency shifts, peak coalescence phenomena, and rapid loss of ion packet coherence, which effectively precludes high-resolution and precision measurements for molecules above ～80-kDa size for a 7-T magnetic field strength. The present approach is based on the summation of many spectra having moderate populations of individual ions and relies on sensitivity sufficient for individual ion detection. While the number of trapped ions contributing to each mass spectrum may generally be insufficient to define the isotopic or charge-state distributions (and thus produce accurate information on the molecular weight distribution in a conventional fashion), the present data processing and summation approach suppresses the noise component (as well as smaller signals) that would otherwise be problematic. Importantly, this approach circumvents natural limitations for very high molecular weight species due to Coulombic interactions and thus provides a basis for much greater resolution and mass measurement accuracy than otherwise possible. This paper presents the details of this approach and its demonstration for the 66-kDa protein bovine serum albumin (where the conventional approach is also feasible) and discusses important aspects of the data manipulation.     

Isotopic peak intensity ratio based algorithm for determination of isotopic clusters and monoisotopic masses of polypeptides from high-resolution mass spectrometric data

Determining isotopic clusters and their monoisotopic masses is a first step in interpreting complex mass spectra generated by high-resolution mass spectrometers. We propose a mathematical model for isotopic distributions of polypeptides and an effective interpretation algorithm. Our model uses two types of ratios: intensity ratio of two adjacent peaks and intensity ratio product of three adjacent peaks in an isotopic distribution. These ratios can be approximated as simple functions of a polypeptide mass, the values of which fall within certain ranges, depending on the polypeptide mass. Given a spectrum as a peak list, our algorithm first finds all isotopic clusters consisting of two or more peaks. Then, it scores clusters using the ranges of ratio functions and computes the monoisotopic masses of the identified clusters. Our method was applied to high-resolution mass spectra obtained from a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer coupled to reverse-phase liquid chromatography (RPLC). For polypeptides whose amino acid sequences were identified by tandem mass spectrometry (MS/MS), we applied both THRASH-based software implementations and our method. Our method was observed to find more masses of known peptides when the numbers of the total clusters identified by both methods were fixed. Experimental results show that our method performed better for isotopic mass clusters of weak intensity where the isotopic distributions deviate significantly from their theoretical distributions. Also, it correctly identified some isotopic clusters that were not found by THRASH-based implementations, especially those for which THRASH gave 1 Da mismatches. Another advantage of our method is that it is very fast, much faster than THRASH that calculates the least-squares fit.     

Quantitative determination of isotope ratios from experimental isotopic distributions

Isotope variability due to natural processes provides important information for studying a variety of complex natural phenomena from the origins of a particular sample to the traces of biochemical reaction mechanisms. These measurements require high-precision determination of isotope ratios of a particular element involved. Isotope ratio mass spectrometers (IRMS) are widely employed tools for such a high-precision analysis, which have some limitations. This work aims at overcoming the limitations inherent to IRMS by estimating the elemental isotopic abundance from the experimental isotopic distribution. In particular, a computational method has been derived that allows the calculation of 13C/12C ratios from the whole isotopic distributions, given certain caveats, and these calculations are applied to several cases to demonstrate their utility. The limitations of the method in terms of the required number of ions and S/N ratio are discussed. For high-precision estimates of the isotope ratios, this method requires very precise measurement of the experimental isotopic distribution abundances, free from any artifacts introduced by noise, sample heterogeneity, or other experimental sources.     

Improving the efficiency of IMS-IMS by a combing technique

A simple method for increasing the efficiency of multidimensional ion mobility spectrometry (IMS-IMS) measurements (as defined by the number of two-dimensional data sets necessary to sample all of the ions in a complex mixture) is illustrated. In this approach, components from a packet containing a mixture of ions are introduced into the first IMS drift region where they are separated based on differences in mobility. At the exit of this region, narrow distributions of ions having identical mobilities are selected, subjected to gentle activation conditions that are intended to induce conformational changes, and transmitted into a second IMS drift region where the new conformations are separated. Here, we describe a simple timing sequence associated with selection and activation of multiple distributions at the entrance of the second drift region in a systematic fashion that improves the efficiency of two-dimensional IMS-IMS by a factor of approximately 8. The method is illustrated by examination of a mixture of tryptic peptides from human hemoglobin.     

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.     

Automated intensity descent algorithm for interpretation of complex high-resolution mass spectra

This paper describes a new automated intensity descent algorithm for analysis of complex high-resolution mass spectra. The algorithm has been successfully applied to interpret Fourier transform mass spectra of proteins; however, it should be generally applicable to complex high-resolution mass spectra of large molecules recorded by other instruments. The algorithm locates all possible isotopic clusters by a novel peak selection method and a robust cluster subtraction technique according to the order of descending peak intensity after global noise level estimation and baseline correction. The peak selection method speeds up charge state determination and isotopic cluster identification. A Lorentzian-based peak subtraction technique resolves overlapping clusters in high peak density regions. A noise flag value is introduced to minimize false positive isotopic clusters. Moreover, correlation coefficients and matching errors between the identified isotopic multiplets and the averagine isotopic abundance distribution are the criteria for real isotopic clusters. The best fitted averagine isotopic abundance distribution of each isotopic cluster determines the charge state and the monoisotopic mass. Three high-resolution mass spectra were interpreted by the program. The results show that the algorithm is fast in computational speed, robust in identification of overlapping clusters, and efficient in minimization of false positives. In approximately 2 min, the program identified 611 isotopic clusters for a plasma ECD spectrum of carbonic anhydrase. Among them, 50 new identified isotopic clusters, which were missed previously by other methods, have been discovered in the high peak density regions or as weak clusters by this algorithm. As a result, 18 additional new bond cleavages have been identified from the 50 new clusters of carbonic anhydrase.     

Use of an artificial immune system derived method for the charge state assignment of small-molecule mass spectra

Knowing the charge state of an ion in a mass spectrum is crucial to being able to assign a formula to it. For many small-molecule peaks in complex mass spectra, the intensities of the isotopic peaks are too low to allow the charge state to be calculated from isotopic spacings, which is the basis of the conventional method of determining the charge state of an ion. A novel artificial intelligence derived method for identifying the charge state of ions, in the absence of any isotopic information or a series of charge states, has been developed using an artificial immune system approach. This technique has been tested against synthetic and real data sets and has proven successful in identifying the majority of multiply charged ions, thereby significantly improving the peak assignment rate and confidence.     

Fine structure in isotopic peak distributions measured using a dynamically harmonized Fourier transform ion cyclotron resonance cell at 7 T

The fine structure of isotopic peak clusters in mass spectra of reserpine and substance P are measured using Fourier transform ion cyclotron resonance mass spectrometry at a 7 T magnetic field. The resolved peaks in the fine structure consist of (13)C, (15)N, (17)O, (18)O, (2)H, (33)S, (34)S, and combinations of them. A recently introduced high-resolution ion cyclotron resonance cell (Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G. J. Am. Soc. Mass Spectrom. 2011, 22, 1125-1133) is used in these experiments. The positions of the experimentally obtained fine structure peaks on the mass scale agree with the isotopic distribution simulations with ≤200 ppb error. Some deviation from the theoretical isotopic distribution is observed, less abundant peaks in the fine structure patterns are a little suppressed compared to the larger ones. We present a method for atomic composition determination using accurate mass data and fine isotopic structure of the mass spectrum. Our method combines the traditional atomic composition determination from accurate mass data by enumeration of all possible formulas within constraints defined a priori with the estimation of element coefficients from resolved isotopic structures. These estimated values allow one to narrow the search space for the composition and therefore to reduce the number of candidate formulas.     

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.     

Optimization of parameters of the linear TOF-SIMS spectrometer by DOE method

Time of flight SIMS spectrometers are widely used in many industrial and scientific applications. Mass spectra of secondary ions ejected from a bombarded surface supply valuable data on its composition and give insights into physical and chemical processes at the surface. The data quality depends on several parameters, which control the spectrometer functioning. This contribution presents our work on an application of a so-called Design of Experiments (DOE) procedure to optimize working parameters of a home-assembled linear TOF-SIMS spectrometer. The efficiency of optimization procedures was tested for mass spectra of positive ions sputtered from Al surface under keV Ar ion bombardment. In particular, we show that use of the method leads to a large increase both of the mass resolution and of the signal/noise ratio (S/N), i.e. improves two quantities, which play a crucial role in mass spectrometry measurements. In addition, it is shown that DOE' method allows to reduce significantly the number of test measurements, and therefore to shorten the adjusting procedure to a minimum.     

Determining the efficiency of a plasma thruster with closed electron drift

We propose a new method for the analysis of the efficiency of ion generation and acceleration, which can be used to determine the ion current in a plasma accelerator (thruster) with closed electron drift. The method is based on the measurement of the energy and angular distributions of ions in the plasma jet, the discharge power, and the jet thrust. The laws of variation of the plasma thruster efficiency as a function of the discharge voltage in the interval of 300–1000 V have been studied.     

The energy spectra of clusters formed during ion sputtering of metals

We propose a method for calculating the energy spectra of neutral and charged clusters with the number of atoms N≤5 sputtered from a metal surface during ion bombardment. The calculated energy distributions of clusters emitted from tantalum and niobium bombarded with atomic gold ions are compared to the published experimental data.     

Collision-induced dissociation spectra obtained by Fourier transform ion cyclotron resonance mass spectrometry using a 13C,15N-doubly depleted protein

Fourier transform ion cyclotron resonance mass spectra of 13C,15N-doubly depleted cystatin A M65L, produced by Escherichia coli grown on 99.9% [12C]glucose and 99.99% [14N]ammonium sulfate, showed salient monoisotopic peaks composed of 12C and 14N. Collision-induced dissociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trapping time in a radio frequency-only hexapole ion guide. Fragment ions in the spectra could be readily assigned to the amino acid sequence, owing to their markedly improved resolution and sensitivity as compared to those with the natural isotopic composition. Detailed analyses of the fragmentation patterns, facilitated by the use of 13C,15N-doubly depleted proteins, enabled the assignment of approximately 180 fragment ions to the sequence, while natural isotopic cystatin A allowed the assignment of approximately 110 fragment ions. Interestingly, no fragmentation was detected between residues 50-61 and 62-67, which are stretches known to be involved in the antiparallel beta-sheet at the center of the protein.     

Stable-isotope dimethyl labeling for quantitative proteomics

In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.     

Improved modelling of the loading spectra using a mixture model approach

In order to perform a fatigue-life analysis of structures the parameters of the structure loading spectra must be assessed. If the load time series are counted using a two-parametric rainflow counting method, the structure loading spectrum provides a probability for the occurrence of a load-cycle with certain amplitude and mean values. It is beneficial for the prediction of the fatigue life to describe the loading spectrum by a continuous function. We have previously discovered that mixtures of Gaussian probability density functions can be used to model the loading spectra. The main problems of this approach that have not been satisfactorily resolved before are related to the estimation of the number of components in the applied mixture models, and to the modelling of the load-cycle distributions with relatively fat tails. In this article, we describe a method for estimating the parameters of mixture models, which allows automatic determination of the number of components in a mixture model. The presented method is applied for modelling simulated and measured loading spectra using mixtures of the multivariate Gaussian or t probability density functions. In the article we also show that the mixture of t probability density functions sometimes better describes the loading spectra than the mixture of Gaussian probability density functions.     

Detection of negative ions from individual ultrafine particles

Aerosol mass spectrometers can be used to classify individual airborne particles on the basis of chemical composition. While positive ion mass spectra are normally used to characterize ultrafine particles (defined here as particles smaller than 200 nm in diameter), negative ion mass spectra can provide complementary information. To effectively utilize the negative ion mass spectra of ultrafine particles, it is important to understand biases in the formation and detection of negative ions. It is found that the intensity of negative ions is generally less than that of positive ions, due to the creation of electrons in the ablation process that must react to form negative ions. The ablation efficiency, defined as the probability that an ablated particle produces a detectable ion signal, exhibits both size and composition dependencies. The ablation efficiency for detection of negative ions follows the same trends as the ablation efficiency for the detection of positive ions: sodium chloride and ammonium nitrate have higher ablation efficiencies than oleic acid, and the ablation efficiency decreases with the particle diameter. The ablation efficiency of negative ions is less than or equal to the ablation efficiency of positive ions, and the relative difference increases as the particle diameter decreases. Pure ammonium sulfate particles exhibit an ablation efficiency too low to be measured in the present experiments. However, trace amounts of sulfate in mixed-composition particles can be readily detected in the negative ion mass spectra.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Surface Coulomb explosions: The influence of initial charge distributions

Using molecular dynamics simulations, we have studied the Coulomb explosion processes on silicon surfaces. Three different initial shapes of ion distributions are used to model the possible localized charge distributions generated in highly charged ion (HCI)-surface impact. The three distinct distributions include a hemispherical, a flat disk, and a long and thin cylindrical geometrical. At t = 0, 100 singly-charged Si ions are embedded in the Si(111) surfaces. In about 100 fs, the strong repulsive electrostatic forces in these three systems cause Coulomb explosions and thus create three different shapes of craters on the surfaces. All simulations are carried to 1.6 ps, at which point the size and shape of the craters are nearly stabilized. The detailed analysis of the ejected ions, atoms, and the substrates reveals the dynamical consequences of the different initial conditions. For these 100 ions, the differences in the total number of the ejected particles, ranging from 245 to 317 particles, appear to be determined by the initial shape of the ionized region and not by the initial repulsive energy restored in the charged region. Contrary to intuition, a long and thin cylindrical distribution is the most efficient pattern for ejecting particles. The underlying mechanism is that ions with this initial configuration transfer more energy to the surrounding atoms. In all three cases, the number of ejected neutral particles are much greater then the number of ions (6–10 times as many atoms as ions). Among the ejected particles, a small percent of particles are found to return the surface at a later time. The angular distribution of ejected particles are also analyzed. While the differences in the distributions of polar angle of the Si atoms of the three configurations is small, the differences in distributions of the ions portray a strong shape dependence in the polar angle.     

Analysis of base oil fractions by ClMn(H2O)+ chemical ionization combined with laser-induced acoustic desorption/fourier transform ion cyclotron resonance mass spectrometry

Laser-induced acoustic desorption (LIAD), combined with chemical ionization with the ClMn(H(2)O)(+) ion, is demonstrated to facilitate the analysis of base oils by Fourier transform ion cyclotron resonance mass spectrometry. The LIAD/ClMn(H(2)O)(+) method produces only one product ion, [ClMn + M](+), for each component (M) in base oils, thus providing molecular weight (MW) information for the analytes. With the exception of one sample, no fragmentation was observed. The mass spectra indicate the presence of homologous series of ions differing in mass by multiples of 14 Da (i.e., CH(2)). All peaks in the spectra correspond to ions with even m/z values and hence are formed from hydrocarbons with no nitrogen atoms, in agreement with the compositional nature of base oils. The MW distributions measured for two groups of base oil samples cover the range 350-600 Da, which is in excellent agreement with the values determined by gas chromatography. Moreover, the hydrocarbon types (i.e., paraffin and cycloparaffins with different numbers of rings) present in each base oil sample can be determined based on the m/z values of the product ions. Finally, the results obtained by using LIAD/ClMn(H(2)O)(+) indicate that the efficiency of the technique (combined desorption and ionization efficiency) is similar for different hydrocarbon types and fairly uniform over a wide molecular weight range, thus allowing quantitative analysis of the base oils. Hence, the product ions' relative abundances were used to determine the percentage of each type of hydrocarbon in the base oil. In summary, three important parameters (MW distributions, hydrocarbon types, and their relative concentrations) can be obtained in a single experiment. This mass spectrometric technique therefore provides detailed molecular-level information for base oils, which cannot be obtained by other analytical methods.