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.     

Ultrahigh-speed calculation of isotope distributions

This paper introduces an ultrahigh-speed algorithm for calculating isotope distributions from molecular formulas, elemental isotopic masses, and elemental isotopic abundances. For a given set of input data (molecular formula, elemental isotopic masses, and elemental isotopic abundances), and assuming round-off error to be negilgible, the new algorithm rigorously produces isotope distributions whose mean and standard deviation are "correct" in the sense that an error-free algorithm would produce a distribution having the same mean and standard deviation. The peak heights are also "correct" in the sense that the height of each nominal isotope peak from the ultrahigh-speed calculation equals the integrated peak area of the corresponding nominal isotope peak from an exact calculation. As a consequence of these properties, the algorithm generally places isotope peaks within millidaltons of their true centroids. The method uses Fourier transform methods and relates closely to two other recently introduced algorithms. The suite of capabilities provided by these three algorithms is sufficient to solve an extremely wide range of problems requiring isotope distribution simulation.     

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.     

Correcting for methane interferences on δ2H and δ18O measurements in pore water using H2Oliquid-H2Ovapor equilibration laser spectroscopy

Cavity ring-down spectroscopy (CRDS) is a new and evolving technology that shows great promise for isotopic δ(18)O and δ(2)H analyses of pore water from equilibrated headspace H(2)O vapor from environmental and geologic cores. We show that naturally occurring levels of CH(4) can seriously interfere with CRDS spectra, leading to erroneous δ(18)O and δ(2)H results for water. We created a new CRDS correction algorithm to account for CH(4) concentrations typically observed in subsurface and anaerobic environments, such as ground waters or lake bottom sediments. We subsequently applied the correction method to a series of geologic cores that contain CH(4). The correction overcomes the spectral interference and provides accurate pore water δ(18)O and δ(2)H values with acceptable precision levels as well as accurate concentrations of CH(4).     

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.     

Oxygen isotopes in nitrite: analysis, calibration, and equilibration

Nitrite is a central intermediate in the nitrogen cycle and can persist in significant concentrations in ocean waters, sediment pore waters, and terrestrial groundwaters. To fully interpret the effect of microbial processes on nitrate (NO3-), nitrite (NO2-), and nitrous oxide (N2O) cycling in these systems, the nitrite pool must be accessible to isotopic analysis. Furthermore, because nitrite interferes with most methods of nitrate isotopic analysis, accurate isotopic analysis of nitrite is essential for correct measurement of nitrate isotopes in a sample that contains nitrite. In this study, nitrite salts with varying oxygen isotopic compositions were prepared and calibrated and then used to test the denitrifier method for nitrite oxygen isotopic analysis. The oxygen isotopic fractionation during nitrite reduction to N2O by Pseudomonas aureofaciens was lower than for nitrate conversion to N2O, while oxygen isotopic exchange between nitrite and water during the reaction was similar. These results enable the extension of the denitrifier method to oxygen isotopic analysis of nitrite (in the absence of nitrate) and correction of nitrate isotopes for the presence of nitrite in "mixed" samples. We tested storage conditions for seawater and freshwater samples that contain nitrite and provide recommendations for accurate oxygen isotopic analysis of nitrite by any method. Finally, we report preliminary results on the equilibrium isotope effect between nitrite and water, which can play an important role in determining the oxygen isotopic value of nitrite where equilibration with water is significant.     

Determination of stable carbon isotopic compositions of low molecular weight dicarboxylic acids and ketocarboxylic acids in atmospheric aerosol and snow samples

We report a new method developed for the determination of stable carbon isotopic composition of homologous alpha,omega-dicarboxylic acids and phthalic acid isolated from environmental samples such as atmospheric aerosols and snow. Dicarboxylic acids are derivatized with BF3/1-butanol to dibutyl esters, which are analyzed for the stable carbon isotopic composition using a capillary GC interfaced to on-line combustion isotope ratio mass spectrometer. The delta13C values for individual dicarboxylic acid are then calculated from delta13C of 1-butanol and butyl ester derivative using a mass balance equation. The accuracy of the delta13C measurement for C2-C10 diacids is within 0.8 per thousand. We report a few examples of the delta13C ratios of saturated C2-C9 alpha,omega-dicarboxylic acids, unsaturated (maleic, phthalic) diacids, and oxocarboxylic acids in the aerosol and snow samples.     

Isotopic Effects on the Temperature of the Triple Point of Water

An investigation into the effects of isotopic composition on the triple point temperature of water has been carried out at the National Institute of Metrology (NIM), China, since redefinition of the kelvin with respect to Vienna Standard Mean Ocean Water (V-SMOW) was officially proposed by the Consultative Committee for Thermometry (CCT) in 2005. In this paper, a comparison of four cells with isotopic analyses and relevant results corrected for isotopic composition, employing the isotope correction algorithm recommended by the CCT, is described. The results indicate that, after application of the corrections, the maximum temperature difference between the cells drops from 0.10 mK to 0.02 mK and that these cells are in good agreement within 0.02 mK. Also, temperature deviations arising from isotopic variations fall in the range from −55.9 μK to + 40.7 μK. We consider that the distillation temperature and degassing time of the production procedure lead to isotopic variations.     

Isotopic analysis of atmospheric formaldehyde by gas chromatography isotope ratio mass spectrometry

Little is known about the isotopic composition of formaldehyde in the atmosphere, a chemical intermediate in hydrocarbon oxidation. Here, we present a promising new method to analyze the carbon (delta 13C) and hydrogen (delta D) isotopic composition of atmospheric formaldehyde. The direct isotopic analytical technique described uses continuous-flow gas chromatography-isotope ratio mass spectrometry, which provides flexibility for either isotopic analysis without correction for derivative functional groups. Current levels of precision of measurement are +/-1.1 and +/-50 per thousand (1 sigma) for delta 13C and delta D analyses, respectively. Concentration of formaldehyde in ambient air is also determined, coincident with isotopic measurement, to a precision of +/-15%. The method has the required sensitivity for analyses of formaldehyde in urban air on relatively small volume grab samples of whole air (10-70L STP), potentially providing high temporal resolution. This is particularly advantageous for studying formaldehyde given its short lifetime and large variability in the atmosphere.     

Counting Fluorescent Dye Molecules on DNA Origami by Means of Photon Statistics

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single‐molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1–20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well‐defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non‐invasively within 150 ms. The method might become a quantifying add‐on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT‐type microscopy.     

A discharge with runaway electrons: Efficient process for the formation of Rydberg states in oxygen molecules

A new method for exciting high-lying states in oxygen molecules is proposed, which is based on the interaction of O₂ molecules with a low-energy (～10²–10⁴ eV) electron beam formed in a discharge with runaway electrons. An algorithm for calculating the electron energy distribution function (EEDF) using the Monte Carlo simulation technique is developed. The EEDF calculation for a discharge in pure oxygen is performed for a plasma pumped with monoenergetic electron beam in the absence of an external electric field. Efficiencies of the formation of Rydberg and Herzberg states in oxygen molecules under conditions of a quasi-stationary dc discharge and a discharge with runaway electrons are compared.     

Generation of O2 from BaSO4 using a CO2-laser fluorination system for simultaneous analysis of delta18O and delta17O

With the observation of mass-independent isotopic anomalies in numerous atmospheric molecules, the ability to measure both delta17O and delta18O in a range of samples is needed. Sulfate oxygen isotopic studies conventionally report only delta18O values. Recent findings indicate that sulfate delta17O and delta18O values, particularly the delta17O value (= delta17O - (0.52)(delta18O)), can provide independent information on the origin, mixing, and transformation of sulfate in the atmospheric and surface environments, which is not resolvable by only delta18O measurements. Existing methods for analyzing sulfate delta17O and delta18O are extremely laborious and demand high-purity BrF5. Here we report a novel method of generating O2 directly from Barite (BaSO4) for simultaneous analysis of delta18O and delta17O by isotope ratio mass spectrometry (IRMS). The method utilizes a CO2-laser fluorination system that can also be used to quantitatively generate O2 from silicates and oxides. Partial but consistent oxygen yields from BaSO4 are obtained for samples >4 mg. Correction factors of +9.4% for delta18O and 4.89% for delta17O are obtained, and there is no deviation in the delta17O value due to the nonquantitative O2 generation. The system may process more than a dozen samples per working day, with analytical error of +/-0.05% and +/-0.8% for delta17O and delta18O, respectively. This new method is ideal for studies emphasizing an accurate sulfate delta17O value.     

Label-Free Identification of Exosomes using Raman Spectroscopy and Machine Learning

Exosomes, nano-sized extracellular vesicles (EVs) secreted from cells, carry various cargo molecules reflecting their cells of origin. As EV content, structure, and size are highly heterogeneous, their classification via cargo molecules by determining their origin is challenging. Here, a method is presented combining surface-enhanced Raman spectroscopy (SERS) with machine learning algorithms to employ the classification of EVs derived from five different cell lines to reveal their cellular origins. Using an artificial neural network algorithm, it is shown that the label-free Raman spectroscopy method's prediction ratio correlates with the ratio of HT-1080 exosomes in the mixture. This machine learning-assisted SERS method enables a new direction through label-free investigation of EV preparations by differentiating cancer cell-derived exosomes from those of healthy. This approach will potentially open up new avenues of research for early detection and monitoring of various diseases, including cancer.     

Functionalized magnetic nanoparticles for small-molecule isolation, identification, and quantification

Functionalized magnetic nanoparticles (MNPs) were synthesized to serve as laser desorption/ionization elements as well as solid-phase extraction probes for simultaneous enrichment and detection of small molecules in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Two laser-absorbing matrices were each conjugated onto MNP to give MNP@matrix which provided high ionization efficiency and background-free detection in MS leading to unambiguous identification of target small molecules in a complex mixture. MNP@matrix was demonstrated to serve as a general matrix-free additive in MALDI-TOF MS analysis of structurally distinct small molecules. Also, MNP@matrix provides a simple, rapid, and reliable quantitative assay for small molecules by mass without either the use of an internal standard or an isotopic labeling tag. Furthermore, the affinity extraction of small molecules from complex biofluid was achieved by probe protein-conjugated MNP@matrix without laborious purification. We demonstrated that a nanoprobe-based assay is a cost-effective, rapid, and accurate platform for robotic screening of small molecules.     

Development and validation of a method to determine the boron isotopic composition of crop plants

We present a comprehensive chemical and mass spectrometric method to determine boron isotopic compositions of plant tissue. The method including dry ashing, a three-step ion chromatographic boron-matrix separation, and (11)B/(10)B isotope ratio determinations using the Cs(2)BO(2)(+) graphite technique has been validated using certified reference and quality control materials. The developed method is capable to determine δ(11)B values in plant tissue down to boron concentrations of 1 mg/kg with an expanded uncertainty of ≤1.7‰ (k = 2). The determined δ(11)B values reveal an enormous isotopic range of boron in plant tissues covering three-quarters of the natural terrestrial occurring variation in the boron isotopic composition. As the local environment and anthropogenic activity mainly control the boron intake of plants, the boron isotopic composition of plants can be used for food provenance studies.     

Conformational stability of adsorbed insulin studied with mass spectrometry and hydrogen exchange.

A new method is described for direct monitoring of the conformational stability of proteins that are physically adsorbed from solution onto a solid substrate. The adsorption-induced conformational changes of insulin are studied using a combination of hydrogen/deuterium (H/D) exchange and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The effect of the surface hydrophobicity on the adsorption-induced conformational changes in the insulin structure is probed by adsorbing insulin on a hydrophilic silica and a hydrophobic methylated silica surface before subjecting the insulin molecules to the isotopic exchange process. The present study describes the experimental procedure of this new application of MALDI. Results show that insulin is more highly and more irreversibly adsorbed to a hydrophobic methylated silica surface than to a hydrophilic silica surface. Hydrogen-exchange experiments clearly demonstrate that the strong interaction of insulin with the hydrophobic surface is accompanied by a strong increase in the H/D-exchange rates and thus in a reduction in the insulin native structural stability. In contrast, H/D-exchange rates of insulin are somewhat reduced upon adsorption on silica from solution.     

An extraction chromatography method for Hf separation prior to isotopic analysis using multiple collection ICP-mass spectrometry

A novel method for the single-step separation of Zr + Hf from all matrix elements of geological samples has been developed for Hf isotopic measurements using multiple collector-ICP-mass spectrometry. The method combines an effective sample decomposition by LiBO2 fusion with a selective separation of Hf + Zr by a solid-phase extraction material based on dipentyl pentyl phosphonate, commercially available as U-TEVA.Spec. Using this simple and rapid procedure, Hf and Zr can be isolated in a single separation step with good recoveries (>90%) and satisfactory blank levels (approximately 55 pg of Hf), so that a subsequent isotopic measurement with ICPMS is possible. An excellent separation from rock-forming constituents is achieved, including those elements (Al, P, Ti, Cr, Fe, Mo, etc.) known to interfere in conventional separation methods based on ion-exchange techniques. The potential of this new method for Hf isotopic analysis is demonstrated by replicate MC-ICPMS measurements of 176Hf/177Hf ratios in seven international reference materials of silicate rocks, spanning a range of Hf contents and bulk compositions.     

Chemical conversion of nitrate and nitrite to nitrous oxide for nitrogen and oxygen isotopic analysis in freshwater and seawater

We present a novel method for nitrogen and oxygen natural isotopic abundance analysis of nitrate and nitrite of seawater and freshwater at environmental concentrations. The method involves the reduction of nitrate to nitrite using spongy cadmium with further reduction to nitrous oxide using sodium azide in an acetic acid buffer. For separate nitrite analysis, the cadmium reduction step is simply bypassed. Nitrous oxide is purged from the water sample and trapped cryogenically using an automated system with subsequent release into a gas chromatography column. The isolated nitrous oxide is then analyzed on a continuous flow isotope ratio mass spectrometer via an open split. This paper describes the basic protocol and reaction conditions required to obtain reproducible natural abundance level nitrogen and oxygen isotopic ratios from nitrate, nitrite, or both, and the results obtained to support these conclusions. A standard deviation less than 0.2 per thousand for nitrogen and 0.5 per thousand for oxygen was found for nitrate samples ranging in concentration from 40 to 0.5 microM (better for nitrite), with a blank of 2 nmol for 50-mL samples. Nitrogen and oxygen isotopic fractionation and oxygen atom exchange were consistent within each batch of analysis. There was no interference from any seawater matrixes. Only one other method published to date can measure the nitrate oxygen isotopic abundance in seawater and none that do so for nitrite alone in the presence of nitrate. This method may prove to be simpler, faster, and obtain isotopic information for lower concentrations of nitrate and nitrite than other methods.