Improved isotopic abundance measurements for high resolution fourier transform ion cyclotron resonance mass spectra via time-domain data extraction

The simultaneous high resolution and accurate mass measurements possible with Fourier transform ion cyclotron resonance mass spectrometry coupled with the gentle ionization of electrospray hold attractions for protein, peptide, and oligonucleotide characterization, including multistage-mass spectrometry measurements for assignment of fragment masses and greater confidence in structural measurements. The detection of cyclotron motion over extended periods of time (in some cases for several minutes) allows higher resolution and mass accuracy. Generally, signal duration has been considered to be limited primarily by background pressure, with ion-neutral collisions leading to the reduction and dephasing of cyclotron motion, causing signal loss. However, recent theoretical work has shown that the ion cloud stability that is a prerequisite for high performance measurements is highly dependent on the electric field generated by the ion cloud, thus giving rise to a minimum number of charges or ions required for extended time-domain signals. The effects of ion population on ion cloud stability and signal duration, and the subsequent effects on resolution and measured isotopic abundances are reported. Individual time-domain signals for bovine insulin isotopic peaks were extracted to allow a comparison of the damping rates for each of these ion clouds and the measured time-domain amplitude maxima are shown to provide a better match with the theoretically predicted isotopic abundances for insulin. These results show that different damping rates of ions of very similar mass, but different ion cloud population sizes, can have dramatic effects on the observed isotopic patterns. Additionally, more accurate, high resolution spectra can be produced by correcting for the effects of the different damping rates that are observed for different ion population sizes.     

Elimination of 13Calpha splitting in protein NMR spectra by deconvolution with maximum entropy reconstruction

Homonuclear 13C-13C couplings can significantly reduce the sensitivity and resolution of multidimensional NMR experiments. The most important of these couplings is the 13Calpha-13Cbeta coupling, and several different methods have been developed to eliminate its effect from spectra used for backbone assignment, including short or constant-time evolution periods, selectively labeled amino acids, and multiple-band decoupling sequences. In this communication we show that postacquisition deconvolution of the spectra with a maximum entropy algorithm can be superior to experimental decoupling. The method is very robust, does not introduce shifts of the resonance positions, and simplifies the measurement of the most important NMR experiments for protein backbone assignment.     

Investigation of site selectivity of the stereoselective deprotonation of cyclohexene oxide using kinetic resolution of isotopic enantiomers in natural abundance

Stereoselective deprotonation of epoxides with lithium amides can occur by abstraction of protons from more than one site. The site selectivity of the deprotonation of cyclohexene oxide by several chiral and achiral lithium amides has been investigated. ²H NMR has been used to measure the relative abundances of the isotopomers of the epoxide containing one deuterium. An isotopic stereoisomer, with deuterium in the site undergoing abstraction, reacts slower than its enantiomer and other isotopomers having protium in the same site due to a kinetic isotope effect. This results in a kinetic resolution yielding a relative excess of the less reactive isotopic stereoisomer. Thus, the relative abundance of such an enantiomer increases when compared with those having protium at the site in question as the reaction proceeds. It can be concluded that deprotonation of cyclohexene oxide using some chiral- and non-chiral lithium amides occurs by β_syn-deprotonation.     

Improved cell typing by charge-state deconvolution of matrix-assisted laser desorption/ionization mass spectra

Robust, specific, and rapid identification of toxic strains of bacteria and viruses, to guide the mitigation of their adverse health effects and optimum implementation of other response actions, remains a major analytical challenge. This need has driven the development of methods for classification of microorganisms using mass spectrometry, particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), that allows high-throughput analyses with minimum sample preparation. We describe a novel approach to cell typing based on pattern recognition of MALDI mass spectra, which involves charge-state deconvolution in conjunction with a new correlation analysis procedure. The method is applicable to both prokaryotic and eukaryotic cells. Charge-state deconvolution improves the quantitative reproducibility of spectra because multiply charged ions resulting from the same biomarker attaching a different number of protons are recognized and their abundances are combined. This allows a clearer distinction of bacterial strains or of cancerous and normal liver cells. Improved class distinction provided by charge-state deconvolution was demonstrated by cluster spacing on canonical variate score charts and by correlation analyses. Deconvolution may enhance detection of early disease state or therapy progress markers in various tissues analyzed by MALDI-MS.     

Some aspects of the high resolution mass spectra of toluene-p-sulphonamides

The cracking patterns of twenty five toluene-p-sulphonamides have been studied. In certain cases an abundant [M ? SO₂] ion is detected: the structural features associated with this phenomenon are discussed.     

Exploring the high-mass components of humic acid by laser desorption ionization mass spectrometry

Leonardite and Elliot soil humic acids have been analyzed by laser desorption ionization mass spectrometry (LDI MS) in the m/z 4000-200,000 range. Positive ion mass spectra for each humic acid obtained under optimum conditions showed a broad high-mass distribution between m/z 20,000 and 80,000. The dependence of the mass distribution on instrumental parameters and solution conditions was used to investigate the nature of the high-mass peaks from humic acid spectra. Our data suggests that macromolecular ions and humic acid aggregates have the same probability of occurrence while cluster ion formation has a low probability of occurrence.     

Band composition analysis: a new procedure for deconvolution of the mass spectra of organometallic compounds

A new chemometric procedure called band composition analysis (BCA) designed for the deconvolution of mass spectra of organometallics is proposed. BCA generates theoretical bands T(i), then combines them to obtain a model band M, which is finally compared with the experimental band E. All of these steps are realized with computer assistance. This modeling yields four parameters characterizing the experimental band: theoretical and model variances s(2) (theor) and s(2) (model), a fit factor alpha and a contribution x(i) from the theoretical band. If s(2) (theor) > 20 the band is deemed complex and needs modeling. The values alpha > 90 indicate that there is good agreement between the experimental and model bands. BCA is particularly effective for the modeling of complex isotopic bands often present in organometallics. Two illustrations of BCA for tetrabutyltin, C(16)H(36)Sn, and 1,1',2,2',3,3'-hexachloroferrocene, C(10)H(4)Cl(6)Fe, are shown.     

Error-systematics of determining simultaneously the isotopic abundance ratios of natural lithium and natural boron as Li2BO2+

Recently, we have shown how the errors delta(j) and delta(k), that occur when measuring the two different isotopic molecular abundance ratios required for analysis, are transformed into the actual errors of elemental isotopic analysis, (deltaEi/Ei)'s. With a view to gain further understanding as to how the errors (deltaEi/Ei)'s are governed, we now evaluate theoretically the effects of selecting different isotopic molecular pairs as the monitor pairs (j and k) for measurements, and of the measurement errors (delta(j) and delta(k)), on the results of analysis (the 6Li/7Li and the 10B/11B abundance ratios), by considering all the constituent elements of Li2BO2+ at their natural isotopic abundances. It is shown that the ratio of measurement errors, delta(j)/delta(k), is a more fundamental parameter than either the individual errors (delta(j) and delta(k)), or their sum, absolute value(delta(j)) + absolute value(delta(k)), in governing deltaEi/Ei. The important implication of this observation is that it reveals the possibility of achieving not only a desired level of accuracy in analysis, but even absolute accuracy (i.e. deltaEi/Ei = 0) by causing mutual cancellation of the effects of individual measurement errors delta(j) and delta(k), through proper regulation of measurement parameters. However, as the measurement errors cannot be pre-set, it is shown how selection of proper monitor pairs (j and k) can help achieve the desired accuracy in analysis. The present work sets guidelines for the more general problem of selecting monitor pairs to avoid larger errors in analysis.     

Characteristics of the mass spectra of acetonides of some natural compounds

Summary The acetonides of alkaloids and coumarins can be identified by their mass-spectrometric properties. The intensity of the fragments characteristic for the dioxolane ring largely depends on the structure of the chain attached to it.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 336–340, May–June, 1972.     

Use of cumulative distribution functions to characterize mass spectra of intact proteins

The MH+ ions of matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) spectra for a series of closely related but otherwise indistinguishable proteins were analyzed for singularity using a distribution free statistic, the Kolmogorov-Smirnov non-parametric statistic, K-S. The approach allows spectra which might otherwise be taken as identical, to be distinguished. Such analysis of the spectra may lead to a greater understanding of the chemistry of the proteins under investigation. The analysis requires only standard instrumentation. A standard data analysis protocol was developed and applied to generate a normalized cumulative distribution function (NCDF) for each spectrum. Differences in the NCDF for two different spectra were calculated and the maximum difference, deltamax compared to critical values of K-S. Values of deltamax exceeding the critical value of K-S are taken as the basis for rejecting the statistical null-hypothesis and assigning statistical significance to the differences in the two spectra. We have shown that this approach allows spectra of 1:1 mixtures of closely related recombinant proteins to be distinguished from either protein alone, and that mixtures of a 45 kDa protein and a labeled version of that protein can be distinguished from the pure material and from one another at the level of about 25%. In addition, we are able to use this approach to characterize the extent to which a synthetic glyococonjugation reaction has proceeded under circumstances of differing reaction times.     

Study and validity of 13C stable carbon isotopic ratio analysis by mass spectrometry and 2H site-specific natural isotopic fractionation by nuclear magnetic resonance isotopic measurements to characterize and control the authenticity of honey

Honey samples were analyzed by stable carbon isotopic ratio analysis by mass spectrometry (SCIRA-MS) and site-specific natural isotopic fractionation measured by nuclear magnetic resonance (SNIF-NMR) to first determine their potentials for characterizing the substance and then to combat adulteration. Honey samples from several geographic and botanical origins were analyzed. The δ¹³C parameter was not significant for characterizing an origin, while the (D/H)_I ratio could be used to differentiate certain single-flower varieties. Application of the official control method of adding a C₄ syrup (AOAC official method 998.12) to our authentic samples revealed anomalies resulting from SCIRA indices that were more negative than −1‰ (permil). A filtration step was added to the experimental procedure and provided results that were compliant with the natural origin of our honey samples. In addition, spiking with a C₄ syrup could be detected starting at 9-10%. The use of SNIF-NMR is limited by the detection of a syrup spike starting only at 20%, which is far from satisfying.     

Fragmentation and rearrangement processes in the high resolution mass spectra of diphenylsilyl compounds

High resolution mass spectra of a series of diphenylsilyl compounds, Ph₂SiX₂(X=H, F, Cl) and Ph₂Si(oy)₂ (Y=Et, Me, H), were recorded. Results from selected metastable defocusing experiments showed that if X or OY can be readily lost stepwise, the relative abundance of the biphenyl radical rearrangement ion (d) is small; if neutral silicon species such as :SiX₂, SiX or :Si(OY)₂ are readily eliminated, the relative abundance of the ion d will be large. The ion d originates from both odd- and even-electron ions, thus making the radical site mechanism, which requires a phenyl radical to polarize the other phenyl group, rather unlikely to be an important general driving force. The condensation of phenyl groups could be more appropriately viewed in terms of bond liability and product stability. In the special case of diphenylsilyl catecholate, in addition to geminal cleavage of phenyl groups, complex fragmentation and rearrangement processes were also observed.     

Electrospray ionization mass spectra of hemoglobin and transferrin by a magnetic sector mass spectrometer. Comparison with theoretical isotopic distributions.

Electrospray ionization mass spectra of human hemoglobin chains and of transferrin were acquired on a magnetic sector mass spectrometer. The observed molecular ion for each hemoglobin chain was in good agreement with the theoretical isotopic distribution at a reasonable resolution of 2000. The clear separation of a variant beta-chain in admixture with the normal counterpart at mass 15,867 that differed from it by 14 Da (0.09%) ensured that a smaller mass difference could be detected. The molecular ions for human transferrin were too broad compared with the theoretical shape to determine the molecular mass accurately, probably due to the heterogeneity of the carbohydrate moiety. A decrease in mass by neuraminidase digestion, however, determined the average number of sialic acids in the molecule.