Deconvolution of mass spectra measured with a non-uniform detector array to give accurate ion abundances

Array detectors are under active development since they offer large improvements in the efficiency of mass spectrum measurements. High quality is always a requirement whether array detectors are used for ions, electrons, UV, x-rays, etc., but in mass spectrometry the very high accuracy frequently needed in ion abundance measurements for isotope ratios is unique. These demands necessitate modelling the measurement process and careful deconvolution of the measured data. A linear model in terms of matrix algebra is presented in which the incident spectrum (unknown) and the measured spectrum are represented by column matrices and the action of the detector array on the incident spectrum is represented by an experimentally measurable square 'A' matrix. Residual noise in this matrix can be minimized following a singular value decomposition procedure and its use in 'forward deconvolution' of measured spectra is discussed. The random error in the incident ion counts is accounted for after the deconvolution since this is not an error from the perspective of the detector. The microchannel plate electron multiplier gain distribution is an important feature of the deconvolution.     

Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. The systematic arrangement of amino acids reported here indicates a possible route toward development of a predictive model for quantitative electron capture/transfer dissociation tandem mass spectrometry, with possible applications in proteomics.     

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH(2)O), Thr (-CH(3)CHO) and Asp (-H(2)O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert.     

Modelling the hydrolysis of succinimide: formation of aspartate and reversible isomerization of aspartic acid via succinimide

In the present study, we have modelled the nucleophilic attack of water and a hydroxyl anion on the carbonyl carbon of a succinimide derivative leading to aspartate and aspartic acid. Calculations have been carried out at the B3LYP/6-31 +G* level in a vacuum. The IEF-PCM methodology has been used to carry out single point calculations in solution. In neutral medium, hydrolysis is facilitated by the presence of a polar continuum, whereas in basic medium the polar environment hinders the hydrolysis of succinimide. The deltaH degrees and deltaS degrees values for the cyclization reactions of aspartic acid yielding succinimide are 29.2 kJ mol(-1) and 133.5 kJ mol(-1) K(-1) respectively in accordance with the experimental results on the isomerization of the Ac-Asp-Gly-NHMe dipeptide unit. In a neutral medium, the isoaspartate: aspartate is found to be 2.2:1 in a vacuum and 3.4:1 in solution, in line with the experimental findings based on the hydrolysis of a tetrapeptide (Ac-Gly-Asn-Gly-Gly-NHMe) and a hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) where this ratio was found to be 3.1:1.     

LC-MS analysis of the formed peptides from N-(O,O-diiso-propyl)phosphoryl aspartic acid

LC-ESI-MS method was used to analyze the formed di-and tri-peptide hi the reaction system of N-(O,O-diisopropyl)phosphoryl aspartic acid and adenosine.Ouster ions of the peptides were given hi the ESI-MS.The structures of these small peptides were confirmed by LC-MS-MS analysis.Compared with the traditional HPLC-UV detection,this method showed good sensitivity and selectivity for peptide in the presence of compounds with strong UV absorption,such as nucle-oside and nucleotide.     

Novel software for data analysis of Fourier transform ion cyclotron resonance mass spectra applied to natural organic matter

Natural organic matter (NOM) occurs as an extremely complex mixture of large, charged molecules that are formed by secondary synthesis reactions. Due to their nature, their full characterization is an important challenge to scientists specializing in NOM as well as analytical chemistry. Ultra‐high‐resolution Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) analysis enables the identification of thousands of masses in a single measurement. A major challenge in the data analysis process of NOM using the FT‐ICR MS technique is the need to sort the entire data set and to present it in an accessible mode. Here we present a simple targeted algorithm called the David Mass Sort (DMS) algorithm which facilitates the detection and counting of consecutive series of masses correlated to any selected mass spacing. This program searches for specific mass differences among all of the masses in a single spectrum against all of the masses in the same spectrum. As a representative case, the current study focuses on the analysis of the well‐characterized Suwannee River humic and fulvic acid (SRHA and SRFA, respectively). By applying this algorithm, we were able to find and assess the amount of singly and doubly charged molecules. In addition we present the capabilities of the program to detect any series of consecutive masses correlated to specific mass spacing, e.g. COO, H₂, OCH₂ and O₂. Under several limitations, these mass spacings may be correlated to both chemical and biochemical changes which occur simultaneously during the formation and/or degradation of large mixtures of compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as α-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an α-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of α-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure.     

Principal component analysis applied to collisionally-activated-decomposition mass spectra of alkylbenzenes

Pattern recognition of the level-1 type is applied to collisionally-activated-decomposition (c.a.d.) mass spectra of eight isomeric C₉H₁₂ alkylbenzenes. Principal component analysis is used for data compression. Only masses with a relative intensity above a variable threshold are selected; no autoscaling is used. Various criteria for deducing the number of factors are compared; because of the error structure, the 3s-misfit and chi-squared criteria are most suitable. The principal components technique produces three factors describing four different groups of c.a.d. mass spectra, those corresponding to n-propyl-, isopropyl-, ethylmethyl- and trimethyl-benzene molecular ions.     

Multilayers quantitative X-ray fluorescence analysis applied to easel paintings

X-ray fluorescence spectrometry (XRF) allows a rapid and simple determination of the elemental composition of a material. As a non-destructive tool, it has been extensively used for analysis in art and archaeology since the early 1970s. Whereas it is commonly used for qualitative analysis, recent efforts have been made to develop quantitative treatment even with portable systems. However, the interpretation of the results obtained with this technique can turn out to be problematic in the case of layered structures such as easel paintings. The use of differential X-ray attenuation enables modelling of the various layers: indeed, the absorption of X-rays through different layers will result in modification of intensity ratio between the different characteristic lines. This work focuses on the possibility to use XRF with the fundamental parameters method to reconstruct the composition and thickness of the layers. This method was tested on several multilayers standards and gives a maximum error of 15% for thicknesses and errors of 10% for concentrations. On a painting test sample that was rather inhomogeneous, the XRF analysis provides an average value. This method was applied in situ to estimate the thickness of the layers a painting from Marco d’Oggiono, pupil of Leonardo da Vinci.     

Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis. Chromatographic data provide a set of sequence-specific information complementary to mass spectrometry. In order to compare measured retention times (RTs) with those calculated from the sequences derived from protein databases, it is necessary to develop chromatographic models and tools allowing the prediction of RT and elution order for peptides with modified residues. In this work we extended recently introduced critical liquid chromatography of biomacromolecule model for prediction of RTs for peptides containing asparagines, aspartic acid, and isoaspartic acid residues.     

Factors affecting immonium ion intensities in the high-energy collision-induced decomposition spectra of peptides

Each amino acid in a peptide has a characteristic immonium ion (H₂N⁺?CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.     

A combined nuclear magnetic resonance and computational study of monohydroxyflavones applied to product ion mass spectra

A method is presented for the estimation of 13C-chemical shifts for carbon atoms in protonated and deprotonated molecules; in principle, this method can be applied to ions in general. Experimental 13C-chemical shifts were found to vary linearly with computed atomic charges using the PM3 method. Pseudo-13C-chemical shifts for atoms in protonated and deprotonated molecules can be estimated from computed atomic charges for such atoms using the above linear relationship. The pseudo-13C-chemical shifts obtained were applied to the rationalization of product ion mass spectra of protonated and deprotonated molecules of flavone and 3-, 5-, 6-, 7-, 2'-, 3'-, and 4'-hydroxyflavones, where product ion formation is due to either cross-ring cleavage of the C-ring (retro-Diels-Alder reaction) or to cleavage of a C-ring bond followed by loss of either a small neutral molecule or a radical. The total product ion abundance ratio of C-ring cross cleavage to C-ring bond cleavage, gamma, varied by a factor of 660 for deprotonated monohydroxyflavones, i.e., from 0.014:1 to 9.27:1. The magnitude of gamma, which is dependent on the relative bond orders within the C-ring of the protonated and deprotonated molecules of monohydroxyflavones, can be rationalized on the basis of the magnitudes of the 13C- and 1H-chemical shifts as determined by nuclear magnetic resonance spectroscopy.     

Conjugation of arginine–glycine–aspartic acid peptides to thermoreversible N‐isopropylacrylamide polymers

Thermoreversible polymeric biomaterials are finding increased acceptance in tissue engineering applications. One drawback of the polymers is their synthetic nature, which does not allow direct interaction of mammalian cells with the polymers. This limitation may be alleviated by grafting arginine–glycine–aspartic acid (RGD) containing peptides onto the polymer backbone to facilitate interactions with cell‐surface integrins. Toward this goal, N‐isopropylacrylamide (NiPAM)‐based thermoreversible polymers containing amine‐reactive N‐acryloxysuccinimide (NASI) groups were synthesized. Conjugation of RGD‐containing peptides to polymers was demonstrated with ¹H NMR spectroscopy and reverse‐phase high‐pressure liquid chromatography. The conjugation reaction was optimal at 4 °C and pH of 8.0, and increased with the increasing NASI content of polymers. With a peptide grafting ratio of 0.25 mol %, there was no significant change in the lower critical solution temperature of the polymers. Finally, the NASI‐containing polymers, cast as films, on tissue culture polystyrene, were shown to conjugate to RGD‐containing peptides and support C2C12 cell attachment. We conclude that NASI‐containing thermoreversible polymers are amenable for grafting biomimetic peptides to impart cell adhesiveness to the polymers. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3989–4000, 2003     

Automatic analysis of mixed spectra : Generalised spectral subtraction applied to electron spin resonance spectroscopy

A new technique is described for estimating the pure component spectra from a set of linearly independent spectra. The process is one of generalised spectral subtraction in which an iterative combination of multivariate linear least-squares analysis and matrix transformation is applied to the input data to give estimates of the number of independent components in the original mixed spectra. This technique is applicable to bipolar data (e.g. from e.p.r. spectra) as well as absorption spectra determined by any spectroscopic technique, provided that the spectra may be reasonably assumed to be an additive mixture of unknown pure components. Numerical model examples are given together with an experimental application to electron spin resonance.     

Kinetic model for reaction of aspartic acid with magnesium carbonate in solution

A kinetic model has been developed for describing the non-catalytic liquid-solid reaction between aspartic acid and magnesium carbonate in solution, with parallel dissolution of aspartic acid particles. The effects of size distribution of both the aspartic acid and magnesium carbonate particles have been accounted for by using a population balance model. The parameters of the model have been estimated by fitting them to experimental data taken from literature.

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Quantitation of ion abundances in fourier transform ion cyclotron resonance mass spectrometry

To improve the analytical usefulness of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), an extensive survey of various methods for quantitation of peak magnitudes has been undertaken using a series of simulated transient response signals with varying signal-to-noise ratio. Both peak height (five methods) and peak area (four methods) were explored for a range of conditions to determine the optimum methodology for quantitation. Variables included dataset size, apodization function, damping constant, and zero filling. Based on the results obtained, recommended procedures for optimal quantitation include: apodization using a function appropriate for the peak height ratios observed in the spectrum (i.e., Hanning for ratios of about 1:10, three-term Blackman-Harris for ratios of ～1:100, or Kaiser-Bessel for ratios of ～1:1000); zero filling until the peaks of interest are represented by 10–15 points (generally obtained with one order of zero filling); and use of the polynomial y=(ax ²+bx+c) ⁿ and the three data points of highest intensity of the peak to locate the peak maximum, Y _max=(?b ²/4a+c) ⁿ . In this peak fitting procedure, which we have termed the “Comisarow method,” n is 5.5, 9.5, and 12.5 for the Hanning, three-term Blackman-Harris, and Kaiser-Bessel apodization functions, respectively. Accuracy of quantitation using an optimal peak height determination is about equal to that for peak area measurements. These recommendations were found to be valid when tested with real FTICR-MS spectra of xenon isotopes.     

Multidimensional statistical analysis applied to electron ionization mass spectra to determine steroid stereochemistry

The differentiation of stereoisomers on the basis of their mass spectra only is usually a difficult challenge even when an informative ionization technique such as electron ionization is used; this is particularly the case for steroids. In this study, multivariate statistical techniques have been applied to the mass spectra of derivatized 5xi-androstane-3xi,17xi-diols (xi = alpha,beta) in order to investigate the possibility of discrimination among the different isomers. After collection of the data from the mass spectra (20 replicates for each of the 8 isomers), each ion was considered as a statistical variable and each mass spectrum as an observation. The more discriminative variables (42 out of the 160 initial ones) were selected using the analysis of variance technique (ANOVA). Thereafter, a linear discriminant analysis (LDA) allowed us to set up a predictive model for stereochemistry determination. The two-dimensional graphical display of the 160 observations on the basis of the canonical variables derived from LDA made it possible to separate the eight isomers. The discrimination of 5alpha and 5beta isomers as well as 3alpha and 3beta was unambiguous, whereas, the discrimination of 17alpha and 17beta epimers was less obvious. The robustness of the model was checked with 40 mass spectra recorded over a 6-month period on different quadrupole mass spectrometers and under different signal acquisition conditions. The percentage of correct assignment of these 'unknown' stereoisomers was 92%; only three 17alpha and 17beta epimers were not correctly plotted in the expected zone. Nevertheless, the performance score was better than those observed with traditional mass spectral libraries. Furthermore, this statistical approach allowed us to identify the main fragment ions involved in the discrimination between isomers: m/z 256 and 421 for isomers 5a-5b; m/z 241 and 331 for isomers 5alpha3alpha-5alpha3beta; m/z 143 and 162 for isomers 5beta3alpha-5beta3beta; and m/z 255 for epimers 17alpha-17beta.     

Two-dimensional correlation spectroscopy techniques applied to ion trap tandem mass spectrometric analysis: nitroaromatics

The technique of two-dimensional (2D) correlation spectroscopy is combined with ion trap tandem mass spectrometric (MS/MS) analysis to provide insight into the energetics and behavior of gas-phase ions. The 2D correlation method is demonstrated by the analysis of the fragment ions produced by the fragmentation of molecular radical cations of nitrobenzene and m-nitrotoluene at a series of applied potentials that effect collision-induced dissociation (CID). The asynchronous correlation intensities show that competitive loss of NO to form [M-NO]+ proceeds at a lower CID energy than loss of NO2 to form [M-NO2]+. The formation of the ion corresponding to [M-NO-CO]+ is shown by the method to occur at a lower CID energy than the formation of [M-NO2]+, but at a higher CID energy than [M-NO]+ for both nitrobenzene and m-nitrotoluene.