Computing fragmentation trees from tandem mass spectrometry data

The structural elucidation of organic compounds in complex biofluids and tissues remains a significant analytical challenge. For mass spectrometry, the manual interpretation of collision-induced dissociation (CID) mass spectra is cumbersome and requires expert knowledge, as the fragmentation mechanisms of ions formed from small molecules are not completely understood. The automated identification of compounds is generally limited to searching in spectral libraries. Here, we present a method for interpreting the CID spectra of the organic compound's protonated ions by computing fragmentation trees that establish not only the molecular formula of the compound and all fragment ions but also the dependencies between fragment ions. This is an important step toward the automated identification of unknowns from the CID spectra of compounds that are not in any database.     

A statistical model for identifying proteins by tandem mass spectrometry

A statistical model is presented for computing probabilities that proteins are present in a sample on the basis of peptides assigned to tandem mass (MS/MS) spectra acquired from a proteolytic digest of the sample. Peptides that correspond to more than a single protein in the sequence database are apportioned among all corresponding proteins, and a minimal protein list sufficient to account for the observed peptide assignments is derived using the expectation-maximization algorithm. Using peptide assignments to spectra generated from a sample of 18 purified proteins, as well as complex H. influenzae and Halobacterium samples, the model is shown to produce probabilities that are accurate and have high power to discriminate correct from incorrect protein identifications. This method allows filtering of large-scale proteomics data sets with predictable sensitivity and false positive identification error rates. Fast, consistent, and transparent, it provides a standard for publishing large-scale protein identification data sets in the literature and for comparing the results obtained from different experiments.     

Automated tandem mass spectrometry by orthogonal acceleration TOF data acquisition and simultaneous magnet scanning for the characterization of petroleum mixtures

The automated acquisition of the product ion spectra of all precursor ions in a selected mass range by using a magnetic sector/orthogonal acceleration time-of-flight (oa-TOF) tandem mass spectrometer for the characterization of complex petroleum mixtures is reported. Product ion spectra are obtained by rapid oa-TOF data acquisition and simultaneous scanning of the magnet. An analog signal generator is used for the scanning of the magnet. Slow magnet scanning rates permit the accurate profiling of precursor ion peaks and the acquisition of product ion spectra for all isobaric ion species. The ability of the instrument to perform both high- and low-energy collisional activation experiments provides access to a large number of dissociation pathways useful for the characterization of precursor ions. Examples are given that illustrate the capability of the method for the characterization of representative petroleum mixtures. The structural information obtained by the automated MS/MS experiment is used in combination with high-resolution accurate mass measurement results to characterize unknown components in a polar extract of a refinery product. The exhaustive mapping of all precursor ions in representative naphtha and middle-distillate fractions is presented. Sets of isobaric ion species are separated and their structures are identified by interpretation from first principles or by comparison with standard 70-eV EI libraries of spectra. The utility of the method increases with the complexity of the samples.     

Detection and quantification of ricin in beverages using isotope dilution tandem mass spectrometry

The toxic plant protein ricin has gained notoriety due to wide availability and potential use as a bioterrorism agent, with particular concern for food supply contamination. We have developed a sensitive and selective mass spectrometry-based method to detect ricin in tap water, 2% milk, apple juice, and orange juice. Ricin added to beverage matrices was extracted using antibody-bound magnetic beads and digested with trypsin. Absolute quantification was performed using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode. The method allows for identification of ricin A chain and B chain and for distinction of ricin from ricin agglutinin within a single analytical run. Ricin-bound beads were also tested for deadenylase activity by incubation with a synthetic ssDNA oligomer. Depurination of the substrate by ricin was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS). This method was used successfully to extract ricin from each beverage matrix. The activity of recovered ricin was assessed, and quantification was achieved, with a limit of detection of 10 fmol/mL (0.64 ng/mL).     

Assay of Sudan I contamination of foodstuff by atmospheric pressure chemical ionization tandem mass spectrometry and isotope dilution

Food safety represents one of the main issues of national and international agencies appointed to health control. In April 2003, a French agency disclosed that powdered or smashed hot chili pepper imported from India and Pakistan was heavily contaminated with a carcinogenic azo dye known as Sudan I. This paper deals with a modern approach for assaying the content of this colorant in foodstuff down to a limit of a few tens of parts per billion. The isotope dilution method combined with APCI tandem mass spectrometry was used. The internal standard, 1-(d5-phenylazo)-2-naphthalenol, was obtained by simple chemistry, and its structure was determined by 1H NMR spectroscopy. The mass spectrometric method is more sensitive than the HPLC approach by a factor of 20.     

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.     

Probability model for assessing proteins assembled from peptide sequences inferred from tandem mass spectrometry data

In shotgun proteomics, tandem mass spectrometry is used to identify peptides derived from proteins. After the peptides are detected, proteins are reassembled via a reference database of protein or gene information. Redundancy and homology between protein records in databases make it challenging to assign peptides to proteins that may or may not be in an experimental sample. Here, a probability model is introduced for determining the likelihood that peptides are correctly assigned to proteins. This model derives consistent probability estimates for assembled proteins. The probability scores make it easier to confidently identify proteins in complex samples and to accurately estimate false-positive rates. The algorithm based on this model is robust in creating protein complements from peptides from bovine protein standards, yeast, Ustilago maydis cell lysates, and Arabidopsis thaliana leaves. It also eliminates the side effects of redundancy and homology from the reference databases by employing a new concept of peptide grouping and by coherently distinguishing distinct peptides from unique records and shared peptides from homologous proteins. The software that runs the algorithm, called PANORAMICS, provides a tool to help analyze the data based on a researcher's knowledge about the sample. The software operates efficiently and quickly compared to other software platforms.     

Automated quantitative analysis of complex lipidomes by liquid chromatography/mass spectrometry

Recent advances in mass spectrometry have revolutionized the analysis of lipid compositions of cells and other biomaterials by simplifying the analytical protocol dramatically and by increasing the sensitivity of detection by several orders of magnitude. However, the throughput of the published mass spectrometric methods is severely limited by data analysis, which requires extensive operator involvement. Consequently, we have developed an automated method that allows unattended identification and quantification of lipid molecular species of all the major lipid classes from a two-dimensional chromatographic/mass spectrometric data set. More than 100 polar lipid species could be automatically quantified from different biological samples with good accuracy and reproducibility. The response was linear over approximately 3 orders of magnitude with the equipment used, and approximately 35 samples could be analyzed in a day. This method makes high-throughput lipidomics feasible in biology, biotechnology, and medicine.     

Direct analysis of bacterial fatty acids by Curie-point pyrolysis tandem mass spectrometry

Although pyrolysis-mass spectrometry (Py-MS) has been used for bacterial taxonomy, many of the mass spectral peaks used for discrimination of organisms have not been correlated to known biomolecules. This work presents the discrimination of five bacterial species based on Py-MS patterns containing only peaks that can be correlated to fatty acids and fatty acid derivatives. These correlations were confirmed by pyrolysis-tandem mass spectrometry of authentic standards and the organisms. The pattern recognition procedures used gave better results when only the fatty acid peaks were used in the analysis than when full spectra were used.     

Quantitative analysis of multiple exocyclic DNA adducts in human salivary DNA by stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry

Exocyclic DNA adducts, including 1,N(2)-propano-2'-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG) and the three lipid peroxidation-related etheno adducts 1,N(6)-etheno-2'-deoxyadenosine (εdAdo), 3,N(4)-etheno-2'-deoxycytidine (εdCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdGuo), play an important role in cancer formation and they are associated with oxidative-stress-induced DNA damage. Saliva is an easily accessible and available biological fluid and a potential target of noninvasive biomarkers. In this study, a highly sensitive and specific assay based on isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) is developed for simultaneous detection and quantification of these five adducts in human salivary DNA. The levels of AdG, CdG, εdAdo, εdCyd, and 1,N(2)-εdGuo, measured in 27 human salivary DNA samples from healthy volunteers, were determined as 104 ± 50, 7.6 ± 12, 99 ± 50, 72 ± 49, 391 ± 198 (mean ± SD) in 10(8) normal nucleotides, respectively, starting with 25 μg of DNA isolated from an average of 3 mL of saliva. Statistically significant correlations were found between levels of εdAdo and εdCyd (γ = 0.8007, p < 0.0001), between levels of εdAdo and 1,N(2)-εdGuo (γ = 0.6778, p = 0.0001), between levels of εdCyd and 1,N(2)-εdGuo (γ = 0.5643, p = 0.0022), between levels of AdG and 1,N(2)-εdGuo (γ = 0.5756, p = 0.0017), and between levels of AdG and εdAdo (γ = 0.3969, p = 0.0404). Only 5 μg of DNA sample was analyzed for simultaneous quantification of these adducts. The easy accessibility and availability of saliva and the requirement for the small amount of DNA samples make this nanoLC-NSI/MS/MS assay clinically feasible in assessing the possibility of measuring 1,N(2)-propano-2'-deoxyguanosine and etheno adducts levels in human salivary DNA as noninvasive biomarkers for DNA damage resulting from oxidative stress and for evaluating their roles in cancer formation and prevention.     

Identification of bacteria using tandem mass spectrometry combined with a proteome database and statistical scoring

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.     

Trace uranium analysis by isotope dilution alpha and mass spectrometry and comparison with other techniques

In this paper uranium determination by isotope dilution mass (MSID) and alpha spectrometry (ASID) using ²³³U as an isotope diluent is discussed. In addition, a new form of MSID employing two tracers (²³⁵U and ²³³U) is developed, where two independent uranium values are obtained for single dilution. The precision and accuracy of each technique are evaluated by comparison with other techniques like X-ray fluorescence and instrumental neutron activation analysis. The techniques, employed here for geological samples, can easily be adapted for environmental materials.     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.     

Analysis of hydrazine in drinking water by isotope dilution gas chromatography/tandem mass spectrometry with derivatization and liquid-liquid extraction

A new isotope dilution gas chromatography/chemical ionization/tandem mass spectrometric method was developed for the analysis of carcinogenic hydrazine in drinking water. The sample preparation was performed by using the optimized derivatization and multiple liquid-liquid extraction techniques. Using the direct aqueous-phase derivatization with acetone, hydrazine and isotopically labeled hydrazine-(15)N2 used as the surrogate standard formed acetone azine and acetone azine-(15)N2, respectively. These derivatives were then extracted with dichloromethane. Prior to analysis using methanol as the chemical ionization reagent gas, the extract was dried with anhydrous sodium sulfate, concentrated through evaporation, and then fortified with isotopically labeled N-nitrosodimethylamine-d6 used as the internal standard to quantify the extracted acetone azine-(15)N2. The extracted acetone azine was quantified against the extracted acetone azine-(15)N2. The isotope dilution standard calibration curve resulted in a linear regression correlation coefficient (R) of 0.999. The obtained method detection limit was 0.70 ng/L for hydrazine in reagent water samples, fortified at a concentration of 1.0 ng/L. For reagent water samples fortified at a concentration of 20.0 ng/L, the mean recoveries were 102% with a relative standard deviation of 13.7% for hydrazine and 106% with a relative standard deviation of 12.5% for hydrazine-(15)N2. Hydrazine at 0.5-2.6 ng/L was detected in 7 out of 13 chloraminated drinking water samples but was not detected in the rest of the chloraminated drinking water samples and the studied chlorinated drinking water sample.     

Ultra performance liquid chromatography isotope dilution tandem mass spectrometry for the absolute quantification of proteins and peptides

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversed-phase column (50 x 2.1 mm i.d., 1.7-microm particle size) with gradient elution at a flow rate of 300 microL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 x 1.0 mm at a flow rate of 30 microL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10-90 fmol/microL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.     

Quantitative method for the analysis of tobacco-specific nitrosamines in cigarette tobacco and mainstream cigarette smoke by use of isotope dilution liquid chromatography tandem mass spectrometry

An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of tobacco specific nitrosamines (TSNA). It utilizes four stable isotope-labeled internal standards instead of two as reported by others. A separate internal standard for each analyte is required to minimize sample matrix effects on each analyte, which can lead to poor analyte recoveries and decreases in method accuracy and precision if only one or two of the internal standards are used, especially for complex sample matrixes and when no sample cleanup steps are performed as in this study. In addition, two ion-transition pairs (instead of one) are used for each analyte for the confirmation and quantification, further enhancing the method's accuracy and robustness. These improvements have led to a new LC-MS/MS method that is faster, more sensitive, and selective than the traditional methods and more accurate and robust than the published LC-MS/MS methods. The linear range of the method was from 0.2 to 250 ng/mL with a limit of detection of each TSNA varied from 0.027 to 0.049 ng/mL. Good correlations between the results obtained by the new method and the traditional method were observed for the samples studied.     

Characterization of hydrophobic peptides by atmospheric pressure photoionization-mass spectrometry and tandem mass spectrometry

The use of photoionization at atmospheric pressure shows great potential for the mass analysis of large apolar or hydrophobic peptides. Mass spectra that were obtained using this technique showed mainly singly charged ions. While polar peptides spectra do not produce fragment ions, others lead to B-type or C-type in-source fragmentation. These dissociation reactions, which could involve electron capture dissociation processes in the case of the C-type ions, are observed for hydrophobic peptides. Both the compatibility of this ionization mode with reversed- or normal-phase liquid chromatographic separation and its sensitivity allow liquid chromatography coupling to both mass spectrometry and tandem mass spectrometry for the analyses of hydrophobic peptide mixtures. Atmospheric pressure photoionization seems to be an interesting alternative method to study hydrophobic peptides that are not easily ionizable by more classical ionization techniques such as electrospray ionization and matrix-assisted laser desorption/ionization.     

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry

Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.     

Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry

Loss of phosphoric acid is the most effective fragmentation reaction of pSer- and pThr-containing phosphopeptides of small size (up to 10-15 residues) in low-energy collision-induced dissociation. Therefore, tandem mass spectrometry with neutral loss scanning was evaluated for its utility to analyze protein phosphorylation using protein kinase A (PKA) catalytic subunit, which is phosphorylated at Thr197 and Ser338, as an example. Analysis of tryptic digests of phosphoproteins by tandem mass spectrometry with scanning for neutral loss of phosphoric acid resulted in spectra with poor signal-to-noise ratio, mainly because of the large size of the phosphopeptides formed (>2 kDa). This unfavorable size was caused by the distribution of tryptic cleavage sites in PKA and by interference of phosphorylation with tryptic cleavage. To generate a set of smaller peptide fragments, digestion was performed using the low-specificity protease elastase. Analysis of the total elastase digest with neutral loss scanning resulted in observation of a set of partially overlapping phosphopeptides with high abundance, providing a complete coverage of PKA phosphorylation sites. The peptide size generated by elastase (0.5-1.5 kDa) is ideally suited for this scan mode, which was found to provide the highest specificity for detection of singly charged phosphopeptides (neutral loss of 98). Identification of the PKA phosphorylation sites was performed by mass spectrometric sequencing of the elastase-derived phosphopeptides, which provided highly informative product ion spectra.