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.     

Exact masses and chemical formulas of individual Suwannee River fulvic acids from ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectra

Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids.     

Versatile online-offline engine for automated acquisition of high-resolution tandem mass spectra

For automated production of tandem mass spectrometric data for proteins and peptides >3 kDa at >50 000 resolution, a dual online-offline approach is presented here that improves upon standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies. An integrated hardware and software infrastructure analyzes online LC-MS data and intelligently determines which targets to interrogate offline using a posteriori knowledge such as prior observation, identification, and degree of characterization. This platform represents a way to implement accurate mass inclusion and exclusion lists in the context of a proteome project, automating collection of high-resolution MS/MS data that cannot currently be acquired on a chromatographic time scale at equivalent spectral quality. For intact proteins from an acid extract of human nuclei fractionated by reversed-phase liquid chromatography (RPLC), the automated offline system generated 57 successful identifications of protein forms arising from 30 distinct genes, a substantial improvement over online LC-MS/MS using the same 12 T LTQ FT Ultra instrument. Analysis of human nuclei subjected to a shotgun Lys-C digest using the same RPLC/automated offline sampling identified 147 unique peptides containing 29 co- and post-translational modifications. Expectation values ranged from 10 (-5) to 10 (-99), allowing routine multiplexed identifications.     

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.     

Increased throughput of proteomics analysis by multiplexing high-resolution tandem mass spectra

High-resolution and high-accuracy Fourier transform mass spectrometry (FTMS) is becoming increasingly attractive due to its specificity. However, the speed of tandem FTMS analysis severely limits the competitive advantage of this approach relative to faster low-resolution quadrupole ion trap MS/MS instruments. Here we demonstrate an entirely FTMS-based analysis method with a 2.5-3.0-fold greater throughput than a conventional FT MS/MS approach. The method consists of accumulating together the MS/MS fragments ions from multiple precursors, with subsequent high-resolution analysis of the mixture. Following acquisition, the multiplexed spectrum is deconvoluted into individual MS/MS spectra which are then combined into a single concatenated file and submitted for peptide identification to a search engine. The method is tested both in silico using a database of MS/MS spectra as well as in situ using a modified LTQ Orbitrap mass spectrometer. The performance of the method in the experiment was consistent with theoretical expectations.     

Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra

Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument. Hence, the sensitivity, throughput and robustness of lipidomics screens were improved without compromising the accuracy and specificity of molecular species identification. The top-down lipidomics strategy lends itself for high-throughput screens complementing ongoing functional genomics efforts.     

Determining plasma isotope composition from high-resolution spectra

Translated from Izmeritel'naya Tekhnika, No. 2, pp. 36–38, February, 1989.     

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.     

Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides

Stable isotope-enriched molecules are used as internal standards and as tracers of in vivo substrate metabolism. The accurate conversion of measured ratios in the mass spectrometer to mole ratios is complicated because a polyatomic molecule containing enriched atoms will result in a combinatorial distribution of isotopomers depending on the enrichment and number of "labeled" atoms. This effect could potentially cause a large error in the mole ratio measurement depending on which isotope peak or peaks were used to determine the ratio. We report a computational method that predicts isotope distributions over a range of enrichments and compares the predicted distributions to experimental peptide isotope distributions obtained by Fourier transform ion cyclotron resonance mass spectrometry. Our approach is accurate with measured enrichments within 1.5% of expected isotope distributions. The method is also precise with 4.9, 2.0, and 0.8% relative standard deviations for peptides containing 59, 79, and 99 atom % excess (15)N, respectively. The approach is automated making isotope enrichment calculations possible for thousands of peptides in a single muLC-FTICR-MS experiment.     

Relative mass defect filtering of high-resolution mass spectra for exploring minor selenium volatiles in selenium-enriched green onions

In this study, the presence of minor Se-containing volatiles in Se-enriched green onions (Allium fistulosum) was investigated using the combination of high-resolution mass spectrometry, inductively coupled plasma mass spectrometry, and a simple relative mass defect-based algorithm to aid trace level analysis of unknown components. This confirmed the structures of volatiles reported previously, along with several unreported small molecular weight Se-containing volatiles from plants, such as MeSeSeSMe. This data analysis technique was also useful to link the results obtained from molecular and elemental mass spectrometry thus aiding in the search for new trace level Se-containing volatiles.     

Appropriate degree of trust: deriving confidence metrics for automatic peak assignment in high-resolution mass spectrometry

Techniques for deriving confidence metrics for the reliability of automatically assigned elemental formulas in complex spectra, from high-resolution mass spectrometers, are described. These metrics can help an analyst to place an appropriate degree of trust in the results obtained from automated spectral analysis of, for example, natural organic materials. To provide these metrics of confidence, common mass spectrometric tests for reliability of peak assignment (mass accuracy/error, relative ion abundance, and rings-plus-double-bonds equivalence) are combined with novel confidence metrics based on the interconnectivity and consistency of a mass difference or mass defect based peak inference network and on the confidence of the initial library matches. These are shown to provide improved peak assignment confidence over manual or simple automatic assignment methods.     

Reduction of the distortion of diffractive-gradient-index high-resolution objectives

The possibility of multiple reductions of the distortion of high-resolution monochromats that consist of diffractive and radial gradient-index lenses is shown. The desirable effect is achieved by mutual compensation of various order members. Design parameters and the field performances of objectives that confirm the effectiveness of a method, are presented.     

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.     

Computation of isotopic peak center-mass distribution by fourier transform

We derive a new efficient algorithm for the computation of the isotopic peak center-mass distribution of a molecule. With the use of Fourier transform techniques, the algorithm accurately computes the total abundance and average mass of all the isotopic species with the same number of nucleons. We evaluate the performance of the method with 10 benchmark proteins and other molecules; results are compared with BRAIN, a recently reported polynomial method. The new algorithm is comparable to BRAIN in accuracy and superior in terms of speed and memory, particularly for large molecules. An implementation of the algorithm is available for download.     

Interpretation of mass spectra from organic compounds in aerosol time-of-flight mass spectrometry

Organic compounds containing a variety of functional groups have been analyzed using aerosol time-of-flight mass spectrometry. Both positive and negative laser desorption/ionization mass spectra have been acquired for compounds of relevance to ambient air particulate matter, including polycyclic aromatic hydrocarbons, heterocyclic analogues, aromatic oxygenated compounds such as phenols and acids, aliphatic dicarboxylic acids, and reduced nitrogen species such as amines. In many cases, positive ion mass spectra are similar to those found in libraries for 70-eV electron impact mass spectrometry. However, formation of even-electron molecular ions due to adduct formation also plays a major role in ion formation. Negative ion mass spectra suggest that organic compounds largely disintegrate into carbon cluster fragments (C(n)- and C(n)H-). However, information about the heteroatoms present in organic molecules, especially nitrogen and oxygen, is carried dominantly by negative ion spectra, emphasizing the importance of simultaneous analysis of positive and negative ions in atmospheric samples.     

Chromatographic purification for the determination of dissolved silicon isotopic compositions in natural waters by high-resolution multicollector inductively coupled plasma mass spectrometry

A procedure is described for accurate Si isotope ratio measurements by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). Dissolved silicon was preconcentrated and separated from other elements present in natural surface waters using anion-exchange chromatography. The optimized procedure provides virtually complete elimination of major inorganic constituents while maintaining Si recovery in excess of 97%. High-resolution capabilities of MC-ICPMS used in this study allow interference-free measurements of 28Si and 29Si isotopes using conventional solution nebulization sample introduction without aerosol desolvation. Owing to the magnitude of polyatomic ion contributions in the region of mass 30, mostly from 14N16O+, measurements of the 30Si isotope can be affected by tailing of the interference signals, making exact matching of analyte and nitric acid concentrations in all measurement solutions mandatory. Isotope abundance ratio measurements were performed using the bracketing standards approach and on-line correction for mass-bias variations using an internal standard (Mg). Uncertainties, expressed as 95% confidence intervals, for replication of the entire procedure are better than +/-0.18/1000 for delta29Si and +/-0.5/1000 for delta30Si. For the first time with MC-ICPMS, the quality of Si isotope abundance ratio measurements could be verified using a three-isotope plot. All samples studied were isotopically heavier than the IRMM-018 Si isotopic reference material.     

Automatic validation of phosphopeptide identifications from tandem mass spectra

We developed and compared two approaches for automated validation of phosphopeptide tandem mass spectra identified using database searching algorithms. Phosphopeptide identifications were obtained through SEQUEST searches of a protein database appended with its decoy (reversed sequences). Statistical evaluation and iterative searches were employed to create a high-quality data set of phosphopeptides. Automation of postsearch validation was approached by two different strategies. By using statistical multiple testing, we calculate a p value for each tentative peptide phosphorylation. In a second method, we use a support vector machine (SVM; a machine learning algorithm) binary classifier to predict whether a tentative peptide phosphorylation is true. We show good agreement (85%) between postsearch validation of phosphopeptide/spectrum matches by multiple testing and that from support vector machines. Automatic methods conform very well with manual expert validation in a blinded test. Additionally, the algorithms were tested on the identification of synthetic phosphopeptides. We show that phosphate neutral losses in tandem mass spectra can be used to assess the correctness of phosphopeptide/spectrum matches. An SVM classifier with a radial basis function provided classification accuracy from 95.7% to 96.8% of the positive data set, depending on search algorithm used. Establishing the efficacy of an identification is a necessary step for further postsearch interrogation of the spectra for complete localization of phosphorylation sites. Our current implementation performs validation of phosphoserine/phosphothreonine-containing peptides having one or two phosphorylation sites from data gathered on an ion trap mass spectrometer. The SVM-based algorithm has been implemented in the software package DeBunker. We illustrate the application of the SVM-based software DeBunker on a large phosphorylation data set.